Methods for Identifying Compounds That Modulate Ion Channel Activity of a Kir Channel

ABSTRACT

Methods for identifying compounds that modulate the ion channel activity of a Kir channel are provided. Methods for identifying compounds that selectively modulate the ion channel activity of specific types of Kir channels based on the turret region of a Kir channel are also provided. Methods for identifying compounds to treat conditions associated with abnormal ion channel activity are also provided. Compounds including purified antibodies and methods of making antibodies which bind to the turret region of a Kir channel are provided. Purified polypeptides including at least a portion of the turret region of a Kir channel and nucleic acid sequences encoding these polypeptides are also provided.

FIELD OF INVENTION

The present invention relates to Kir channel proteins and methods foridentifying compounds that modulate ion channel activity by Kirchannels. In particular, the present invention relates to identifyingcompounds which are useful for treating diseases related to the functionof Kir channel proteins.

BACKGROUND OF THE INVENTION

Inward rectifier K+ charnels (Kir channel proteins) are involved in thecontrol of many physiological processes that are important to humanhealth. Kir channel proteins normally function as K+ (potassium)selective pores that span cell membranes. The Kir channels are referredto as inward rectifier K+ (Kir) channels based on a fundamental ionconduction property of these channels: given an equal but oppositeelectrochemical driving force K+ conductance into the cell far exceedsconductance out of the cell.

Among their many functions Kir channel proteins control the pace of theheart, regulate secretion of hormones into the blood stream, generateelectrical impulses underlying information transfer in the nervoussystem and control airway and vascular smooth muscle tone. It isbelieved that various disease states are directly related to thefunction of Kir channel proteins. Members of this channel family includeKir1-Kir7, (Kubo et al., Pharmacological Rev., 57:509-526, 2005)Hypertension, atrial fibrillation, and type 2 diabetes are related toKir channel protein function and are serious conditions for which newtherapies are needed. Specific links between Kir channel proteins anddisease have been found. Kir1.1 channels are present in the kidney andregulate salt secretion into the urine. Heritable mutations involvingKir1.1 cause Barter's syndrome and hypotension. Compounds whichselectively inhibit Kir1.1 have the potential to serve as a new form ofanti-hypertensive agent in which hypokalemia, a major side-effect ofcurrently used diuretics, should in principle not be a problem. Thus,hypertensive individuals could benefit from Kir1.1 inhibitor-basedtherapies. Kir3.1 and Kir3.4 channels, which assemble to form aheteromultimer, are called G-protein-gated K+ channels (GIRK). Thesechannels control heart rate through stimulation by the parasympatheticnervous system. GIRK channel knock-out mice do not develop atrialfibrillation under any of the usual stimuli that induce this arrhythmiain mice. (Claphan et al., JACC 37, 2136-2143 (Jun. 15, 2001))Accordingly, inhibition of GIRK channels in humans might provideeffective treatment for atrial fibrillation. Kir6 channels are expressedin beta cells of the pancreas and control insulin secretion. With theidentification of compounds that selectively inhibit the Kir6 channelnew therapies could be realized for the treatment of type 2 diabetes.Accordingly, Kir channel proteins are good targets for the treatment ofvarious diseases.

The Kir channel family of proteins are very similar to each other inboth sequence and, by inference, structure; thus, it has been verydifficult to identify compounds that can specifically modulate one kindof Kir channel protein without cross-reacting with other types of Kirchannel proteins.

For the first time the structure of a eukaryotic Kir channel has beendetermined, and a structural feature “the turret region” has beenidentified that is highly ordered in structure and, based on the aminoacid sequences will differ among Kir channels. Prior to this structure,only the structure of a prokaryotic Kir channel had been determined.(Nishida et al., EMBO, vol. 26, pp. 4005-4015 (2007)) The turret is animportant functional region of the protein and faces the outside of thecell making this region an attractive target for identifying potentialtherapeutic compounds. Given the identification of the turret region inthe various Kir channel proteins and the structure in a prototype, thepresent invention provides a variety of methods by which the turretregion may be used to identify compounds having therapeutic utility fortreating the various diseases related to the function of Kir channels.

The present invention provides for the first time the expression andpurification of a eukaryotic Kir channel as explained in detail below.Study of the structure of this eukaryotic Kir channel resulted in arealization of the importance of the turret region and the invention ofmethods which allow identification of therapeutic compounds that canselectively bind to different members of the Kir channel family ofproteins.

SUMMARY OF THE INVENTION

The present invention relates to methods for identifying a compound thatmodulates ion channel activity of a Kir channel including identifying acompound which binds the turret region of a Kir channel; and determiningif the compound modulates ion channel activity of the Kir channel.

In particular embodiments, the method may be used to identify anantibody that binds the turret region of a Kir channel and modulates theKir channel's activity. The antibody may be human, chimeric orhumanized. The antibody may also be a polyclonal antibody, monoclonalantibody, an intact immunoglobulin molecule, an antibody fragment, ascFv, a Fab, a F(ab)2, a Fv, or a disulfide linked Fv.

In another embodiment, the method may be used to identify suitablenucleic acid molecules that can modulate a Kir channel's activity bybinding to its turret region. In such an embodiment, the nucleic acidmay be a DNA or RNA molecule. In certain embodiments the nucleic acid isan aptamer. The method may also include identifying a suitable nucleicacid by using in vitro selection techniques.

In another embodiment, the method is used to identify a protein orpeptide that can bind a turret region of a Kir channel and modulate theKir channel. In this embodiment, the protein/peptide may be attached toa protein scaffold or displayed on the surface of a phage.

In another embodiment, the method discussed above is used to screen forsmall molecules that can modulate Kir channel activity by binding to theKir channel's turret region.

In any of the methods discussed above, the Kir channel may be a humanKir channel or a chicken/human hybrid Kir channel. Typically, thechicken/human hybrid Kir channel will comprise a human Kir channelturret region.

Various standard biochemical assays may be used to identify whether acompound binds to the turret region of a Kir channel in the method ofthe present invention. For example, the identifying step may comprise anELISA and a Western blot to determine if the compound binds to aproperly folded Kir channel but not to a denatured Kir channel.Moreover, the identifying step may comprise determining if the compoundbinds to a Kir channel with a normal turret region but not a mutatedturret region.

Regarding the determining whether a compound modulates the activity of aKir channel, various electrophysiological assays may be used such astwo-electrode voltage clamp, patch clamp, and planar lipid bilayerassays. Alternatively or additionally, the determining step may includea fluorescent assay such as one utilizing a thallium specificfluorescent dye.

In another aspect, the present invention relates to a method foridentifying a compound that selectively modulates ion channel activityof a specific type of Kir channel including identifying a compound whichbinds the turret region of a specific type of Kir channel but does notbind to other types of Kir channels; and determining if the compoundmodulates the activity of the Kir channel.

In another embodiment, the present invention relates to a method ofidentifying a compound to treat a condition associated with abnormal ionchannel activity by a Kir channel including identifying a compound whichbinds the turret region of a Kir channel; determining if the compoundmodulates ion channel activity of the Kir channel; and administering thecompound which modulates ion channel activity to a subject to determineif the compound is able to treat the condition. In such a method, thecondition may be diabetes mellitus, hypertension, cardiac arrhythmia, orepilepsy.

In another aspect, the present invention relates to a purified antibodythat specifically binds to an epitope in the turret region of a Kirchannel. In this embodiment, the purified antibody may be a polyclonalantibody, a monoclonal antibody, an intact immunoglobulin molecule, anantibody fragment, a scFv, a Fab, a F(ab)2, a Fv, or a disulfide linkedFv. The antibody may specifically bind to a human Kir channel such as aKir1, Kir2, Kir3, Kir4, Kir5, Kir6, or Kir7 channel. The antibodypreferably binds to an epitope within the turret region of a human Kirchannel such as Kir1.1, Kir1.2, Kir2.1, Kir2.2, Kir2.3, Kir2.4, Kir3.1,Kir3.4, Kir4.1, Kir4.2, Kir5.1, Kir6.1, or Kir6.2 channel. Even morepreferably, the antibody binds to the variable portion of the turretregion of a human Kir channel.

In another embodiment, the present invention relates to a method ofmaking an antibody that specifically binds to an epitope in the turretregion of a human Kir channel, including providing a chicken/humanhybrid Kir channel, wherein the chicken/human hybrid comprises a humanKir channel turret region; immunizing a non-human animal with thechicken/human hybrid Kir channel; and determining whether the antibodyis binding to the human Kir channel turret region. In this embodiment,the chicken portion of the chicken/human hybrid Kir channel may bederived from a chicken Kir2.2 channel. Moreover, in this embodiment, thehuman Kir channel turret region may be derived from Kir1, Kir2, Kir3,Kir4, Kir5, Kir6, or Kir7. Preferably, the human Kir channel turretregion is derived from a human Kir1.1, Kir1.2, Kir2.1, Kir2.2, Kir2.3,Kir2.4, Kir3.1, Kir3.4, Kir4.1, Kir4.2, Kir5.1, Kir6.1, or Kir6.2channel.

In another embodiment, the present invention relates to a method ofmaking an antibody that specifically binds to an epitope in the turretregion of a human Kir channel, including providing a human Kir channel;immunizing a non-human animal with the Kir channel; and determiningwhether the antibody is binding to the human Kir channel turret region.,

In another embodiment, the present invention relates to a purifiedpolypeptide that consists of the turret region of human Kir channelssuch as Kir1.1, Kir1.2, Kir2.1, Kir2.2, Kir2.3, Kir2.4, Kir3.1, Kir3.4,Kir4.1, Kir4.2, Kir5.1, Kir6.1, or Kir6.2. In another aspect, thepresent invention relates to an isolated nucleic acid comprising anucleotide sequence that encodes a polypeptide that consists of theturret region of human Kir channels such as Kir1.1, Kir1.2, Kir2.1,Kir2.2, Kir2.3, Kir2.4, Kir3.1, Kir3.4, Kir4.1, Kir4.2, Kir5.1, Kir6.1,or Kir6.2.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1C shows key residues in eukaryotic Kir channels. A sequencealignment of chicken Kir2.2 (GI: 118097849), human Kir2.2 (GI:23110982),human Kir2.1 (GI:8132301), human Kir1.1 (GI:1352479), human Kir3.1(GI:1352482), human Kir3.4 (GI:1352484), human Kir6.1 (GI:2493600),human Kir7.1 (GI:3150184), KirBac1.1 (GI:33357898), KcsA (GI:39654804),and rat Kv1.2 (G1:73536156) is shown. For all the Kir sequences only thecore region corresponding to the expressed protein and atomic structureof Kir2.2 is included in the alignment. For Kv1.2 only the transmembranepore region is shown. Secondary structure elements are indicated abovethe sequences and the turret is shown in small dotted lines above thesequence. Residues discussed in the text are boxed in a series ofalternating dashes and dots (acidic residues), a series of large dots(two disulfide-bonded cysteines), alternating dashes and pairs of dots(the inner helix bundle activation gate), series of small dashes(conserved residues among the turrets of eukaryotic Kir channels), aseries of small dots (the selectivity filter and E139), and a series oflarge dashes (critical residues for channel-PIP₂ interactions).

FIG. 2A-2E illustrates a structure of Kir2.2. (FIG. 2A) Stereoview of aribbon representation of the Kir2.2 tetramer from the side with theextracellular solution above. Four subunits of the channel are shown.Approximate boundaries of the lipid bilayer are shown as bars. (FIG. 2B)A close-up view of the pore-region of a single subunit (in ribbonrepresentation) with the turret, pore helix and selectivity filterlabeled. Side chains of residues E139, R149 and a pair ofdisulfide-bonded cysteines (C123 and C155) are shown as sticks. Ionizedhydrogen-bonds are indicated by dashed black lines. The region flankedby the two disulfide-bonded cysteines is stippled. (FIG. 2C) Electrondensity (wire mesh, 2F_(o)-F_(c), calculated from 50-3.1 Å using phasesfrom the final model and contoured at 1.0σ) is shown for the side chainsof E139 and R149 forming a salt-bridge. (FIG. 2D) (FIG. 2E) K⁺selectivity filter of the Kir2.2 channel (FIG. 2 D) compared with thatof the Kv1.2-Kv2.1 paddle chimera channel (FIG. 2E, PDB ID 2R9R). Forclarity, only two of the four subunits are shown. K⁺(cross hatchedcircles), water molecules (solid spheres), and hydrogen bonds betweenR149 and E139 (Kir, dashed black lines), or between D379, M380 andwaters (Kv, dashed black lines) are shown,

FIG. 3A-3G illustrates the cavity and gates region of a Kir channel.(FIG. 3A) (FIG. 3B) Electron density in the cavity of the Kir2.2 channel(A, F_(o)-F_(c) omit map, calculated from 50-3.1 Å using phases from thefinal model and contoured at 2.0σ) and of the KcsA channel (FIG. 3B, PDBID 1K4C, F_(o)-F_(c) omit map, calculated from 50-3.1 Å using phasesfrom the final model and contoured at 2.8σ). The channels are shown asribbon representations with the subunit closest to the viewer removed.Only the side chains facing the cavity are shown (sticks). (FIG. 3C)(FIG. 3D) Comparison of the transmembrane inner helix bundle activationgate of Kir2.2 (FIG. 3C) with the KcsA structure (FIG. 3D, PDB ID 1K4C).For clarity, only two of the four subunits (ribbon) are shown. Sidechains of the residues in the bundle crossing are shown as sticks andvan der Waals surfaces. K⁺ ions are shown as cross hatched spheres.Inner and Outer helices are indicated. (FIG. 3E) Superposition of thechicken Kir2.2 cytoplasmic domain (α-carbon trace) and the mouse Kir2.1cytoplasmic domain (α-carbon trace, PDB ID 1U4F) in stereo viewed fromthe extracellular side. (FIG. 3F) (FIG. 30) Comparison of the apex(G-loop) of the cytoplasmic pores of Kir2.2 (FIG. 3F) and mouse Kir2.1(FIG. 3G), with the same view as FIG. 3E. The cytoplasmic domains areshown as α-carbon traces, with residues 303-309 (Kir2.2) and 302-308(Kir2.1) shown as CPK models.

FIG. 4A-4F illustration of ion binding sites. (FIG. 4A) (FIG. 4B) (FIG.4C) Electron density (wire mesh) of Rb⁺ (FIG. 4A, F_(o)-F_(c) mapcalculated to 4.0 Å, contoured at 3.5σ for density in the filter and2.0σ for density elsewhere), Sr²⁺ (FIG. 4B, 10 mM, F_(o)-F_(c) mapcalculated to 3.3 Å, contoured at 1.5σ for density in the cavity and3.0σ for density elsewhere) and Eu³⁺ (FIG. 4C, 10 mM, anomalousdifference map calculated to 6.0 Å, contoured at 2.8σ) inside the Kir2.2channel ion conduction pathway. Kir2.2 is represented as a α-carbontrace with the transmembrane domain and cytoplasmic domain closest toviewer removed for clarity. The ions are shown as spheres. (FIG. 4D)Electron density (200 mM Sr²⁺, F_(o)-F_(c) map calculated from 50-3.8 Å,contoured at 2.5σ, wire mesh) of Sr²⁺ (spheres) in the cavity of Kir2.2.The channel is shown as a ribbon with the subunit closest to the viewerremoved. Only the side chains facing the cavity are shown (sticks).(FIG. 4E) Stereoview of the ion binding site near the upper ring ofcharges in the cytoplasmic domain of Kir2.2, viewed from theextracellular side. Residues E225, H227, E300, and Q311 are shown assticks, and hydrogen bonds between them are indicated as dashed blacklines. Electron density (200 mM Sr²⁺, F_(o)-F_(c) map calculated from50-3.8 Å, contoured at 4.5σ) of Sr²⁺ (spheres) is shown as wire mesh.(FIG. 4F) Stereoview of the ion binding site at the lower ring ofcharges in the cytoplasmic domain of Kir2.2, viewed from theintracellular side. Residues F255, D256, and K257 are shown as sticks,and hydrogen bonds between D256 from different subunits are indicated asdashed black lines. Electron density (200 mM Sr²⁺, F_(o)-F_(c) mapcalculated from 50-3.8 Å, contoured at 4.5 a) of Sr²⁺ (spheres) is shownas wire mesh.

FIG. 5A-5D illustrates the unique structure of the extracellularentryway. (FIG. 5A) (FIG. 5B) Surface representation of chicken Kir2.2(FIG. 5A) and Kv1.2-Kv2.1 paddle chimera (FIG. 5B, PDB ID 2R9R) instereo, viewed from the extracellular side. The four protrusions formedby the top of the turrets are highlighted with a black perimeter andF148 in Kir2.2 is labeled. (FIG. 5C) Stereo representation of electrondensity (wire mesh) for the turret region (2F_(o)-F_(c), calculated from50-3.1 Å using phases from the final model and contoured at 1.0σ). Theturret is shown as sticks (colored according to atom types), andresidues corresponding to the highlighted protrusions in panel A arelabeled. (FIG. 5D) A close-up view of the turret region in a singlesubunit in stereo. Side chains of those conserved residues among theturrets of eukaryotic Kir channels, as well as C155 are shown as sticks.Hydrogen bonds between H108, D110 and C123 are indicated as dashed blacklines.

FIG. 6A-6D results showing the chicken Kir2.2 channel is a strong inwardrectifier. (FIG. 6A) (FIG. 6B) Macroscopic currents are shown from anuninjected oocyte (FIG. 6A) and a chicken Kir2.2 channel injected oocyte(FIG. 6B) without subtracting leak and capacitive currents. The currentswere recorded from oocytes using two-electrode voltage-clamp. Voltagepulses: holding potential (h.p.) 0 mV, depolarizing steps: −80 mV to +80mV, ΔV=10 mV, stepping back to 0 mV. (FIG. 6C) Macroscopic currentsrecorded from oocytes using patch-clamp. The three current traces show acurrent trace recorded on-cell (labeled B), a trace recorded immediatelyafter excision of the inside-out patch (labeled C), and a trace recordedapproximately 10 minutes after the excision (labeled A) Voltage pulses:ramp from −80 mV to =80 mV over 10 seconds duration, (FIG. 6D) I-V curvefrom a patch containing only a few channels. The single channel currentis graphed as a function of voltage (inset).

FIG. 7 provides a surface representation of Kir2.2, viewed from the sidewith the extracellular side above. The surface is shaded for qualitativeassessment of the negative and positive electrostatic potential at thesurface.

FIG. 8 illustrates ion binding sites of Kir2.2 in the selectivityfilter, central cavity, upper and lower rings of charges are shown assticks (oxygens and stippled). The channel is represented as a a-carbontrace with the transmembrane domain and cytoplasmic domain closest toviewer removed for clarity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based in part on the discovery of an importantstructural feature present in Kir channel proteins. in particular, thepresent invention relates to the discovery of a “turret region” which ishighly ordered in structure and which differs in sequence amongdifferent Kir channel proteins. In addition, this turret region facesthe outside of the cell making the protein accessible to compounds thatbind or otherwise interact with this turret region thereby affecting theability of the Kir channel to function. The discovery of the fact thatthis turret region, which differs in sequence among members of the Kirchannel family, is structured provides a basis to identify compoundswhich can treat disease states related to Kir channel functions.

Example 1 provided below presents a determination of the crystalstructure of a eukaryotic Kir channel protein. In particular, thecrystal structure of the chicken Kir channel protein, Kir2.2 ispresented. Excluding unstructured amino and carboxy termini, the chickenKir2.2 protein is 90% identical to human Kir2.2. More importantly, forthe purposes of the present invention, these structural studiesdemonstrate that Kir channels have a large structured turret regionwhich provide the basis for the development of compounds that may beused to bind and interact with these turrets and treat disease statesrelated to the functioning of Kir channels. In particular, these turretregions suggest approaches to the development of inhibitory compoundswhich will bind to a specific member of the Kir channel family ofproteins and inhibit Kir channel function.

The turret region of a variety of Kir channel proteins are identified inFIGS. 1A-1C and in the sequence listings of the present application. Inparticular, FIGS. 1A-1C illustrates that a sequence alignment of varioushuman Kir channels indicates that the turret region begins with aconsensus sequence HGDL (or minor sequence variations thereof) andextends six amino acid residues after a highly conserved cysteineresidue labeled as C123 in FIGS. 1A-1C. This turret region is highlyconserved and most of the variation that occurs in the sequence islocated in a variable portion located after the sequence HGDL up to thecysteine labeled as C123. This variable portion within the turret regionconstitutes a basis for differentiating Kir channels from one anotherand provides a target for mutagenesis assays to identify compoundscapable of turret specific binding.

Given the identification of the structured turret region in the crystalstructure, the turret region of other Kir channels may be identifiedusing sequence alignment programs and the teachings of the presentinvention relating to the consensus sequence and structural features ofthe Kir channels.

Based on this structural information, methods are presented below inwhich the identification of the turret region and knowledge of the aminoacid sequence of the turret may be used to develop assays to identifytherapeutic compounds which include, but are not limited to, antibodies,nucleic acids, peptides and small molecules that are capable ofselective binding to Kir channel proteins.

In general the methods of the present invention for identifying acompound that modulates ion channel activity of a Kir channel comprisesa two step process: a first step of identifying a compound which bindsthe turret region of a Kir channel; and a second step of determining ifthe compound modulates the ion channel activity of the Kir channel.

Production of Antibodies

In a first method for identifying compounds that modulate the ionchannel activity of a Kir Channel, antibodies are prepared against a Kirchannel. A variety of Kir channels are known and the methods describedbelow may be used to prepare antibodies against any Kir channel.

Given the present discovery of the importance of the turret region indistinguishing one Kir channel from another, it is particularly usefulto obtain antibodies which bind the turret region,

The Antigens and Assay Reagents

Two types of Kir channel proteins may be of particular utility inpreparing antibodies. The first type are human Kir channel proteins. Thesecond type are chimeric constructs which utilize a non-human sequence,preferably a eukaryotic sequence, such as a chicken sequence, inparticular a chicken Kir 2.2 sequence into which a human Kir channelturret region has been inserted, thereby replacing the native turretregion. As an example, chimeric proteins which utilize a chicken Kir2.2“scaffold” into which the turret region from a given human Kir channelis inserted may be used to prepare antibodies which are specific fordifferent human Kir channel proteins. Both human and chimeric Kirchannels may be full length proteins or may contain deletions at theamino and/or carboxy termini of the protein if desired.

Conventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art may be used in order to preparehuman and chimeric Kir channel proteins. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds.(1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins,eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel et al,(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994); Molecular Cloning: A Laboratory Manual Third Edition [JosephSambrook and David W, Russell Cold Spring Harbor Laboratory Press(2001)]; The Condensed Protocols from Molecular Cloning: A LaboratoryManual [Joseph Sambrook and David W. Russell, Cold Spring HarborLaboratory Press (2006)]; Gene Cloning and Manipulation Second Edition[Christopher Howe, Cambridge University Press (2007)].

The cDNA sequences for exemplary human Kir channels are presented in SEQID NOS 30-43. The cDNA sequence of the chicken Kir2.2 channel ispresented in SEQ ID NO: 45. DNA and cDNA sequences for other types ofKir channels are available in public databases, The turret regions ofexemplary human Kir proteins are identified in SEQ ID NOs: 46-56.

Expression of Chicken Kir 2.2

As an example of expression and purification of a eukaryotic Kir channela protocol for preparing a chicken Kir 2.2 channel protein is providedbelow. Using standard techniques this procedure may be modified toprepare any of the human Kir channel proteins or a desired chimeric Kirchannel protein.

To prepare the chicken Kir 2.2 channel, a synthetic gene fragment (BioBasic, Inc.) encoding residues 38 to 369 of chicken Kir2.2 channel(GI:118097849) was ligated into the XhoI/EcoRI cloning sites of amodified pPICZ-B vector (Invitrogen). The resulting protein has greenfluorescent protein (GFP) and a 1D4 antibody recognition sequence(TETSQVAPA) on the C terminus (I), separated by a PreScission proteasecleavage site (SNSLEVLFQ/GP).

The construct was linearized using PmeI and transformed into a HIS+strain of SMD1163 of Pichia pastoris (Invitrogen) by electroporation(BioRad Micropulser). Transformants were selected on YPDS platescontaining 400-1200 μ/ml Zeocin (Invitrogen). Resistant colonies weretested for expression by anti-1D4 tag Western Blot. For large-scaleexpression, small cultures grown from the best expressing colony werediluted into BMGY media (Invitrogen) and inoculated at 29° C. overnight,until OD600 reached between 20-30. Cells were then pelleted, resuspendedin BMM media (Invitrogen) and expressed overnight at 24° C. Cells wereharvested, flash-frozen in liquid N2, and stored at −80° C. untilneeded.

Cells were lysed in a Retsch, Inc. Model MM301 mixer mill (5×3.0 minutesat 25 cps). The lysis buffer contained 150 mM KCl, 50 mM TRIS-HCl pH8.0, 0.1 mg/ml deoxyribonuclease I, 0.1 μg/ml pepstatin, 1 μg/mlleupeptin, 1 μg/ml aprotinin, 0.1 mg/ml soy trypsin inhibitor, 1 mMbenzamidine, 0.1 mg/ml AEBSF, with 1 mM phenylmethysulfonyl fluorideadded just before lysis (3.0 ml lysis buffer/g cells). pH of the lysatewas adjusted to 8.0 with KOH. The lysate was extracted with 100 mM DM(n2 decyl-β-D-maltopyranoside, Anatrace, solgrade) at room temperaturefor 1 hour with stirring, and then centrifuged for 40 minutes at 30,000g, 10° C. Supernatant was added to 1D4-affinity resin pre-equilibratedwith 150 mM KCl, 50 mM TRIS-HCl pH 8.0, and 4 mM DM. Suspension waslayered with Argon and mixed by inversion for 2 hours at roomtemperature. Beads were collected on a column by gravity, washed with 2column volumes of buffer (150 mM KCl, 50 mM TRIS-HCl pH 8.0, 1 mM EDTApH 8.0, and 4 mM DM), and eluted with buffer plus 1 mg/ml 1D4 peptide(AnaSpec, Inc.) over 1 hour at room temperature. 20 mM DTT(Dithiothreitol) and 3 mM TECP were added to eluted protein. The proteinwas then digested with PreScission protease (20:1 w/w ratio) overnightat 4° C. Concentrated protein was further purified on a Superdex-200 gelfiltration column in 150 mM KCl, 20 mM TRIS-HCl pH 8.0, 4 mM DM(anagrade), 3 mM TCEP, 20 mM DTT and 1 mM EDTA at 4° C. In a preferredembodiment, the protein extract is maintained in a mild detergent, suchas DM, which will maintain the three-dimensional structure of the Kirchannel.

Preparation of Human/Chicken Hybrid Kir Channels

Using standard techniques in molecular biology, chimeric Kir channelprotein may be prepared by inserting the turret region of a human Kirchannel protein into the Kir2.2 chicken sequence described above. Thelocation of the turret regions are identified in FIGS. 1A-1C.

By way of example, site-directed mutagenesis procedures may be used toinsert the coding sequence for a human turret region into a eukaryotic“scaffold” Kir channel coding region. In a preferred embodiment,Strategene's QuickChange® is used. QuickChange® utilizes a supercoileddouble-stranded DNA (dsDNA) vector with an insert of interest and twosynthetic oligonucleotide primers containing the desired mutation. Theoligonucleotide primers, each complementary to opposite strands of thevector, are extended during temperature cycling by PfuTurbo DNApolymerase. The desired mutation (in this case—the insertion of thehuman turret region) should be in the middle of the primer with about10-15 bases of correct sequence on both sides. Incorporation of theoligonucleotide primers generates a mutated plasmid containing staggerednicks. Following temperature cycling, the product is treated with Dpn I.The Dpn I endonuclease (target sequence: 5′-Gm⁶ATC-3′) is specific formethylated and hemimethylated DNA and is used to digest the parental DNAtemplate and to select for mutation-containing synthesized DNA. DNAisolated from almost all E. coli strains is Dam methylated and thereforesusceptible to Dpn I digestion. The nicked vector DNA containing thedesired mutations is then transformed into XL1-Blue supercompetentcells. See, e.g. U.S. Pat. Nos. 5,789,166, 5,932,419, and 6,391,548.

As an example, the chicken Kir2.2 protein may be used as a scaffold andthe human Kir2.2 channel turret region synthesized for insertion. Thismethodology can be repeated with any combination of scaffold protein andhuman turret region.

Preparation of Mutated Turret Regions

Given the identification of the turret regions in the human Kir channelssite directed mutagenesis or other techniques known in the art may beused to prepare proteins having mutations in the DNA sequence of theturret. In a preferred embodiment, Strategene's QuickChange® is used.Such mutations should be non-silent mutations—that is the mutationsshould result in amino acid changes at positions within the turretregion.

Generation of Antibodies

A human Kir protein or a chimeric Kir channel protein is prepared usingstandard techniques such as those outlined herein, and used in standardtechniques to obtain antibodies.

A variety of antibodies may be used in the present invention and suchantibodies include but are not limited to polyclonal, monoclonal, human,humanized chimeric, an intact immunoglobulin molecule, an antibodyfragment, single chain, ScFv, Fab fragments, F(ab)₂ Fab, Fv and adisulfide linked Fv.

Various procedures known in the art may be used for the production ofantibodies. Host animals can be immunized by injection with a human Kirchannel protein, or chimeric Kir protein or fragments of these proteins.Animals which may be used to generate antibodies include, but are notlimited to, rabbits, mice, rats, sheep, goats, and others known in theart. The human and chimeric proteins of the present invention may alsobe conjugated to an immunogenic carrier, e.g., bovine serum albumin(BSA) or keyhole limpet hemocyanin (KLH). Various adjuvants may be usedto increase the immunological response, depending on the host species,including but not limited to Freund's (complete and incomplete), mineralgels such as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hernocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacteriumparvum.

For preparation of monoclonal antibodies directed toward a Kir channelprotein of the present invention, any technique that provides for theproduction of antibody molecules by continuous cell lines in culture maybe used, These include but are not limited to the hybridoma techniqueoriginally developed by Kohler and Milstein [Nature 256:495-497 (1975)],as well as the trioma technique, the human B-cell hybridoma technique[Kozbor et al., Immunology Today 4:72 1983); Cote et al., Proc. Nail.Acad. Sci. U.S.A. 80:2026-2030 (1983)], and the EBV-hybridoma techniqueto produce human monoclonal antibodies [Cole et al., in MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)]. Inaddition, techniques developed for the production of “chimericantibodies” [Morrison et al., J. Bacterial. 159:870 (1984); Neuberger etal., Nature 312:604-608 (1984); Takeda et al., Nature 314:452-454(1985)] by splicing the genes from a mouse antibody molecule specificfor an isolated Kir channel protein of the present invention, orconserved variants thereof, together with a fragment of a human antibodymolecule of appropriate biological activity can be used.

Human antibodies can be prepared using any technique. Examples oftechniques for human monoclonal antibody production include thosedescribed by Cole et al, (Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, p. 77, 1985) and by Boemer et al, (J. Immunol., 147(1):86-95,1991). Human antibodies (and fragments thereof) can also be producedusing phage display libraries (Hoogenboom et al., J. Mol. Biol.,227:331, 1991; Marks et al., J. Mol, Biol., 222:581, 1991), Informationon monoclonal and other types of therapeutic antibodies can also befound in Cellular and Molecular Immunology, 6th Edition, [A. K. Abbas,A. H. Lichtman, S. Pillai (Saunders Elsevier Press, 2007)], and U.S.Pat. Nos. 7,390,887 and 7,629,171. For a discussion of various types oftherapeutic antibodies, see Strategies and Challenges for the NextGeneration of Therapeutic Antibodies, A. Beck, T. Wurch, C. Bailly andN. Corvaia, Nature Rev. Immuno. 10, 345-352 (2010).

Human antibodies can also be obtained from transgenic animals. Forexample, transgenic, mutant mice that are capable of producing a fullrepertoire of human antibodies, in response to immunization, have beendescribed (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA,90:2551-255 (1993); Jakobovits et al., Nature, 362:255-258 (1993);Bruggennann et al., Year in Immunol., 7:33 (1993)).

Humanized antibodies may also be used in the present invention. Antibodyhumanization techniques generally involve the use of recombinant DNAtechnology to manipulate the DNA sequence encoding one or morepolypeptide chains of an antibody molecule. Accordingly, a humanizedform of a non-human antibody (or a fragment thereof) is a chimericantibody or antibody chain (or a fragment thereof, such as an Fv, Fab,Fab′, or other antigen-binding portion of an antibody) which contains aportion of an antigen binding site from a non-human (donor) antibodyintegrated into the framework of a human (recipient) antibody. Methodsfor humanizing non-human antibodies are well known in the art. Forexample, humanized antibodies can be generated according to the methodsof Winter and co-workers (Jones et al., Nature, 321:522-525 (1986),Riechmann et al., Nature, 332:323-327 (1988), Verhoeyen et al., Science,239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences forthe corresponding sequences of a human antibody. Methods that can beused to produce humanized antibodies are also described in U.S. Pat. No.4,816,567 (Cabilly et al.), U.S. Pat. No. 5,565,332 (1-Hoogenboom etal.), U.S. Pat. No. 5,721,367 (Kay et al.), U.S. Pat. No. 5,837,243 (Deoet al.), U.S. Pat. No. 5,939,598 (Kucherlapati et al.), U.S. Pat. No.6,130,364 (Jakobovits et al.), and U.S. Pat. No. 6,180,377 (Morgan etal.).

Techniques described for the production of single chain antibodies [U.S.Pat. Nos. 5,476,786 and 5,132,405 to Huston; U.S. Pat. No. 4,946,778]can be adapted to produce single chain antibodies specific for a Kirchannel protein. An additional embodiment of the invention utilizes thetechniques described for the construction of Fab expression libraries[Huse et al., Science 246:1275-1281(1989)] to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificityfor the Kir channel proteins.

Antibody fragments which contain the idiotype of the antibody moleculecan be generated by known techniques. For example, such fragmentsinclude but are not limited to: the F(ab′)₂ fragment which can beproduced by pepsin digestion of the antibody molecule; the Fab′fragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragment, and the Fab fragments which can be generated bytreating the antibody molecule with papain and a reducing agent.

Antibodies or fragments thereof, whether attached to other sequences ornot, can also include insertions, deletions, substitutions, or otherselected modifications of particular regions or specific amino acidsresidues, provided the activity of the antibody or antibody fragment isnot significantly altered or impaired compared to the non-modifiedantibody or antibody fragment. Such methods are readily apparent to askilled practitioner in the art and can include site-specificmutagenesis of the nucleic acid encoding the antibody or antibodyfragment.

Nucleic Acids

The compounds of the present invention include nucleic acids. Inparticular, nucleic acid sequences capable of binding to a Kir channelmay be used in the practice of the present invention. These nucleicacids may be identified using in vitro selection of sequences which bindKir channel proteins, in particular the turret region of the Kir channelproteins. One type of nucleic acid that is of particular interest is anaptamer. Typically aptamers are small nucleic acid sequences rangingfrom 15-50 bases in length that fold into defined secondary and tertiarystructures that bind to another molecule. This binding is not thetypical nucleic acid to nucleic acid hydrogen bond formation but thebinding of aptamers can include all other types of covalent andnoncovalent binding. In a preferred embodiment, the nucleic acid is DNA,however, other nucleic acids such as RNA may be used. The nucleic acidsmay be modified or prepared using techniques known in the art toincrease the stability of nucleic acids. Representative examples of howto make and use aptamers to bind a variety of different target moleculescan be found in the following U.S. Pat. Nos. 5,582,981; 5,595,877;5,637,459; 6,020,130; 6,028,186; 6,030,776; and 6,051,698. See alsoPublished U.S. patent application Ser. No. 11/917,884 (publication No.US2009/0155779A1); Bock L C, Griffin L C, Latham J A, Vermaas E H, TookJ J (February 1992). “Selection of single-stranded DNA molecules thatbind and inhibit human thrombin” Nature 355(6360): 564-6; Bunka D H,Stockley P G (August 2006) “Aptamers come of age—at last” Nat RevMicrobio. 4(8): 588-96.

Small Protein/Peptide Compounds

Small Protein/Peptide Compounds may also be used in the practice of thepresent invention. In particular, small proteins may be prepared andscreened for the ability to bind to a Kir channel protein based onbinding assays disclosed herein and known in the art. Small moleculessuch as toxins may also be used in the practice of the invention. Inparticular, small proteins/peptides modeled on toxins which bind to Kirchannel proteins may be prepared and tested for the ability to bind andmodulate the activity of Kir channel proteins. (Ramu, et al. (2008)Engineered specific and high affinity inhibition for a subtype of inwardrectifier Kir channels Proc. Nat'l Acid Sci USA 105:10774-10778)

A variety of toxins may provide information useful in designing acompound useful in the practice of the present invention. In particular,scorpion toxins (Lu and Mackinnon, 1997 Biochemistry, vol. 36, no, 23,pp 6936 to 6940) snake toxins (for example, the 57 amino acidδ-dendrotoxin from the green mamba snake which inhibits Kir 1.1channels, (J. P. Imredy, C. Chen, R. Mackinnon, BioChemistry 37, 14867(Oct. 20, 1998)) and bee venom toxins (Ramu, et al. (2008) Engineeredspecific and high affinity inhibition for a subtype of inward rectifierKir channels Proc. Nat'l Acid Sci USA 105:10774-10778) may be helpful insynthesizing libraries of protein/peptide compounds that can bind andeffect a Kir channel. Known toxins are often small proteins typicallybetween 20 and 50 amino acids in size containing disulfide bridges. Forsome of these toxins, the surface important for binding to a Kir channelprotein is known and stretches of amino acids of less than 10 aminoacids are believed to be important for binding specificity.

A library of these toxin-based compounds may be prepared whilemaintaining the key amino acids such as cysteine residues that areimportant for the folding and structure of the proteins. The amino acidresidues important for binding to a Kir channel may be randomized togenerate proteins/peptides with enhanced binding strength and turretbased specificity for the different members of the Kir channel family ofproteins.

Phage Display

One method known in the art to rapidly screen a large variety ofpotential binding proteins/peptides is a phage display assay.

Phage display libraries may be prepared using known protocols.“Filamentous fusion phage: novel expression vectors that display clonedantigens on the virion surface”. Science 288 (4705): 1315-1317. Smith GP, Petrenko V A (1997). “Phage display”. Chem. Rev. 97 (2): 391-410.Kehoe J W, Kay B K (2005), “Filamentous phage display in the newmillennium”. Chem. Rev. 105 (11): 4056-4072. Hufton S E, Moerkerk P T,Meulemans E V, de Brüine A, Arends J W, Hoogenboom H R (1999). “Phagedisplay of cDNA repertoires: the pVI display system and its applicationsfor the selection of immunogenic ligands.” J. Immunol. Methods 231(1-2): 39-51, Lunder M, Bratkovic T, Doljak B, Kreft S, Urleb U,Strukelj B, Plazar N. (2005). “Comparison of bacterial and phage displaypeptide libraries in search of target-binding motif”. Appl. Biochem.Biotechnol. 127 (2): 125-31. Lunder M, Bratkovic T, Kreft S, Strukelj B(2005). “Peptide inhibitor of pancreatic lipase selected by phagedisplay using different elution strategies”. J. Lipid Res. 2005 46 (7):1512-6.)

Using phage display the protein/peptide constructs may be expressed onthe outer coat of the phage. To identify useful sequences a Kir channelprotein may be immobilized on the surface of a well of a standard assayplate, and a phage that displays a protein that binds to Kir channelswill bind the target Kir channel protein and remain bound whilenon-binding phage are removed by washing the plates. The bound phage canbe eluted and used to produce more phage for further binding assays.These binding assays may be performed with Kir channels having mutatedturrets and wild type turrets to select for proteins/peptides that bindin the turret region. Repeated cycles of these binding assays(‘panning’) results in the identification of phage containingpotentially strong binding sequences.

Phage that contain these strong binding sequences can be used to infecta suitable bacterial host, and phagemids are collected and the DNAsequence of interest encoding the binding region excised and sequencedto identify the protein/peptide compound which be further tested usingthe assays described below.

Small Molecules

Small molecules may also be used in the practice of the presentinvention. In particular, small molecules may be prepared and screenedfor the ability to bind to a Kir channel protein based on binding assaysdisclosed herein and known in the art. See for example U.S. Pat. No.6,641,997. Additionally, small molecule libraries may also be screened.

Immunoassays

Once an antibody has been generated by the methods described above, avariety of different immunoassays may be performed to identifyantibodies that bind property folded Kir channels, are specific for theturret region of the Kir channel and can differentiate between differentmembers of the Kir family based on the turret region.

It is believed that ELISA and Western blot assays are straightforwardand efficient assays to identify such antibodies before performingfunctional assays such as electrophysiological assays.

In general, immunoassays involve contacting a Kir channel protein withan anti-Kir channel antibody under conditions effective, and for aperiod of time sufficient, to allow the formation of immune complexes(primary immune complexes). Forming such complexes is generally a matterof simply bringing into contact the antibody and the Kir channel proteinsample and incubating the mixture for a period of time long enough forthe antibodies to form immune complexes with, i.e., to bind to, anymolecule (e.g., antigens) present to which the antibodies can bind.

In many forms of immunoassay, the sample-antibody composition, such asan ELISA plate or Western blot, can then be washed to remove anynon-specifically bound antibody species, allowing only those antibodiesspecifically bound within the primary immune complexes to be detected.Immunoassays can include methods for detecting or quantifying the amountof a molecule of interest (such as the disclosed biomarkers or theirantibodies) in a sample. In general, the detection of an immunocomplexformation is well known in the art and can be achieved by numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any radioactive, fluorescent, biological orenzymatic tags or any other known label. Such assays include but are notlimited to ELISA, western blots, radioimmunoassay, (enzyme-linkedimmunosorbant assay), “sandwich” immunoassays, immunoradiometric assays,gel diffusion precipitin reactions, immunodiffusion assays, in situimmunoassays (using colloidal gold, enzyme or radioisotope labels, forexample), precipitation reactions, agglutination assays (e.g., gelagglutination assays, hemagglutination assays), complement fixationassays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, and other assays known in the art.

Antibody binding can be detected by detecting a label on the primaryantibody or the primary antibody is detected by detecting binding of asecondary antibody or reagent to the primary antibody. For some assays,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention.

The use of immunoassays to detect a specific protein can involve theseparation of the proteins by electrophoresis. Electrophoresis is themigration of charged molecules in solution in response to an electricfield. Their rate of migration depends on the strength of the field; onthe net charge, size and shape of the molecules and also on the ionicstrength, viscosity and temperature of the medium in which the moleculesare moving. As an analytical tool, electrophoresis is simple, rapid andhighly sensitive. It is used analytically to study the properties of asingle charged species, and as a separation technique. Electrophoresisis used in the Western blots described below.

ELISA

Enzyme-Linked Immunosorbent Assay (ELISA), or more generically termedEIA (Enzyme ImmunoAssay), is an immunoassay that can detect an antibodyspecific for a protein. In such an assay, a detectable label bound toeither an antibody-binding or antigen-binding reagent is an enzyme. Whenexposed to its substrate, this enzyme reacts in such a manner as toproduce a chemical moiety which can be detected, for example, byspectrophotometric, fluorometric or visual means. Enzymes which can beused to detectably label reagents useful for detection include, but arenot limited to, horseradish peroxidase, alkaline phosphatase, glucoseoxidase, galactosidase, ribonuclease, urease, catalase, malatedehydrogenase, staphylococcal nuclease, asparaginase, yeast alcoholdehydrogenase, alpha.-glycerophosphate dehydrogenase, triose phosphateisomerase, glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. For descriptions of ELISA procedures, see Voller,A. et al., J. Clin. Pathol. 31:507-520 (1978); Butler, J. E., Meth.Enzymol. 73:482-523 (1981); Maggio, E. (ed.), Enzyme Immunoassay, CRCPress, Boca Raton, 1980; Butler, J. E., In: Structure of Antigens, Vol.1 (Van Regenmortel, M., CRC Press, Boca Raton, 1992, pp. 209-259;Butler, J. E., In: van Oss, C. J. et al., (eds), Immunochemistry, MarcelDekker, Inc., New York, 1994, pp. 759-803; Butler, J. E. (ed.),Immunochemistry of Solid-Phase Immunoassay, CRC Press, Boca Raton,1991); Crowther, “ELISA: Theory and Practice,” In: Methods in MoleculeBiology, Vol. 42, Humana Press; New Jersey, 1995; U.S. Pat. No.4,376,110, each of which is incorporated herein by reference in itsentirety and specifically for teachings regarding ELISA methods.

In preferred embodiments of the present invention, ELISA assays are usedto identify antibodies that bind to the Kir channel proteins and arespecific to the turret region.

As illustrated in FLOWCHART I, a first ELISA assay is performed toidentify antibodies that bind to the Kir channel protein. As an example,if a human Kir 2.2 channel protein is used as an antigen, an ELISA Assayis performed to identify antibodies that bind to human Kir 2.2 channelprotein.

By way of an example ELISA assay, solutions are prepared as follows:

-   -   Buffer A: Protein buffer containing detergent slightly above CMC    -   Coating Solution—Buffer A+20 ug/ml Protein (50 ul/well, 5.0        ml./plate)    -   Wash Solution—Buffer A (200 ul/well×14 washes; 2.8 ml/well, 280        ml total/plate)    -   Blocking Solution—Buffer A+5% BSA (0.45u filtered) (400 u/well,        40 ml./plate)    -   Primary Ab solution—Cell culture supernatant (diluted 1:1 with        2×Buffer A/2% BSA) or control sera (1:100 in Buffer A+2% BSA)    -   Secondary Ab Solution—1:10,000 goat α mouse-Horseradish        peroxidase conjugate in Buffer A+2% BSA (100ul/well, 10        ml/plate)

Substrate Solution—1:1 TMB:H₂O₂ (100 ul/well, 10 ml/plate)

-   -   Stop Solution—2M H2SO4 (100 ul/well, 10 ml./plate)        The following steps are then performed:

a) Add 50 ul of coating solution to each well. Prepare coated plates theday before the assay and store at 4° C. overnight, or prepare on day ofassay and allow to shake for 1 hour at room temperature. Add solutiondirectly to the bottom of the well, avoiding the sides as much aspossible. Coat at least 2 more wells than you have samples for (+) and(−) controls, Leave at least 2 wells uncoated (just wash solution) asnegative controls.

b) Remove coating solution by pouring out and smacking plate face downon a paper towel. Wash wells 3× by adding 200 ul of wash solution toeach well, shaking for 1 minute, pouring out wash solution and smackingplates face down on paper towels.

c) Add 300 ul of blocking solution to each well and let plates sit atroom temperature for 2 hour.

d) Remove blocking solution and wash wells 3× with 200 ul of washingsolution.

e) Add 50 ul 2×Buffer to each well (except controls)

f) Add 50 ul primary antibody solution to the 50 ul 2×buffer in eachwell and mix. Add diluted (+) control serum to a coated and uncoatedwell and diluted (−) control serum to coated and uncoated well. Letplates sit at room temperature for 1 hour on orbital shaker.

g) Remove Primary Ab solution and wash wells 3× with 200 ul washingsolution.

h) Add 100 ul secondary Ab solution to each well and let plates sit atroom temperature for 1 hour.

The plates are then examined to determine if antibodies for a Kirchannel protein are present using standard techniques as describedabove.

Western Blot

Western blotting or immunoblotting allows the determination of themolecular mass of a protein and the measurement of relative amounts ofthe protein present in different samples. Detection methods includechemiluminescence and chromagenic detection. Standard methods forWestern blot analysis can be found in, for example, D. M. Bollag et al.,Protein Methods (2d edition 1996) and E. Harlow & D. Lane, Antibodies, aLaboratory Manual (1988), U.S. Pat. No. 4,452,901, each of which isherein incorporated by reference in their entirety for teachingsregarding Western blot methods. Generally, proteins are separated by gelelectrophoresis, usually SDS-PAGE. For the assays used in the presentinvention for initial antibody screening, it is preferred to use anSDS-PAGE so as to denature the Kir channel proteins used in the blot.The proteins are transferred to a sheet of special blotting paper, e.g.,nitrocellulose, though other types of paper, or membranes, can be used.The proteins retain the same pattern of separation they had on the gel.The blot is incubated with a generic protein (such as milk proteins) tobind to any remaining sticky places on the nitrocellulose. An antibodyis then added to the solution which is able to bind to its specificprotein.

The attachment of specific antibodies to specific immobilized antigenscan be readily visualized by indirect enzyme immunoassay techniques,usually using a chromogenic substrate (e.g. alkaline phosphatase orhorseradish peroxidase) or chemiluminescent substrates. Otherpossibilities for probing include the use of fluorescent or radioisotopelabels (e.g., fluorescein, ¹²⁵I). Probes for the detection of antibodybinding can be conjugated anti-immunoglobulins, conjugatedstaphylococcal Protein A (binds IgG), or probes to biotinylated primaryantibodies (e.g., conjugated avidin/streptavidin).

As illustrated in FLOWCHART 1, a western blot is performed to determineif an antibody binds to the denatured form of a Kir channel protein, Thecombination of the ELISA and the Western blot Kir channel assays asillustrated in FLOWCHART 1 facilitates the identification of antibodiesthat recognize the properly folded native Kir channel (ELISA Positive)but not the denatured (Western Negative) form of the protein.

In particular, if a given antibody binds to a Kir channel protein in anELISA assay (“ELISA Positive”), but fails to bind to the same Kirchannel protein in a Western blot (Western Negative), then the antibodyis binding to the native conformation of the protein but not thedenatured form.

Identification of Antibodies with Turret Specificity

Given the discovery in the present invention of the importance of theturret regions and the identification of the region of the Kir proteinswhich constitute the turret region it is possible to prepare Kir channelproteins which contain mutations located in the turret region. This inturn provides the basis for identification of antibodies that arespecific for the turret region of the Kir channel proteins. Inparticular, the determination of the atomic structure of whatconstitutes the turret region of the Kir channels identifies where tointroduce such mutations so as to selectively identify anti-Kir Channelantibodies that are directed against the turret. Such mutations may bemade in a variety of places, such as following the L residue in thesequence HGDL (or slight variations of that sequence) and up to but notincluding the conserved cysteine labeled C123 in the structure of theproteins (see FIGS. 1A-1C). Examples of these variable portions ofcertain turret regions is provided in SEQ ID NOs 1-13. Kir channelproteins which contain mutations in the turret region, or preferably inthe variable portion, may be used in assays described below.

To identify antibodies that are specific for the turret region of theKir channel further ELISA assays may be performed as illustrated inFLOWCHART 1. These ELISA assays utilize Kir channels with mutated turretregions. Antibodies that bind a normal Kir channel in an ELISA assay butdo not bind a channel with a mutated turret will be isolated since theseantibodies may be considered turret specific—that is the epitope for theantibody is located in the turret region of the protein. The source ofthese antibodies will be used to prepare monoclonal antibodies usingstandard techniques as described above. An additional assay describedbelow and presented in FLOWCHART 2 identifies antibodies or othercompounds with the ability to bind the turret region using a fluorescentassay.

Assays for Kir Channel Activity

Even if an antibody or other type of compound binds the turret regionits utility as a therapeutic compound is based on its functional effecton a Kir channel, Accordingly, the next step is to determine if anantibody which binds the turret region of a Kir channel is capable ofmodulating electrolyte processing. Monoclonal antibodies prepared fromturret specific antibodies identified above can be used inelectrophysiological assays as can other compounds found to have bindingspecificity for the turret region of Kir channels.

There are a variety of electrophysiological assays known to those withskill in the art which may be used to determine whether the compounds ofthe present invention have an effect on the electrophysiological stateof a Kir Channel.

Useful electrophysiological assays include a variety of in vitro and invivo assays, e.g., measuring voltage, current, measuring membranepotential, measuring ion flux, e.g., potassium or rubidium, measuringpotassium concentration, measuring second messengers and transcriptionlevels, and using e.g., voltage-sensitive dyes, radioactive tracers,electrode voltage clamps and patch-clamp electrophysiology. Such assayscan be used to test for both inhibitors and activators of Kir channels.

Modulators of the Kir channels may be tested using biologically active,functional Kir channels, either recombinant or naturally occurring. Inrecombinantly based assays, the subunits are typically expressed andmodulation is tested using one of the in vitro or in vivo assaysdescribed herein.

In brief, samples or assays that are treated with a potential Kirchannel turret binding compounds inhibitors or activators are comparedto control samples without the test compound, to examine the extent ofmodulation. Control samples e.g. those untreated with the compounds areassigned a relative Kir channel activity value of 100. Inhibition ispresent when Kir channel activity value relative to the control is about90%, preferably 50%, more preferably 25%.

It should be noted that the compounds may also result in activation ofKir channels. Activation of channels is achieved when the select Kirchannel activity value relative to the control is 110%, more preferably150%, more preferable 200% higher. It is possible that for treating somediseases states such activating compounds may be useful alone or incombination with inhibitors.

Changes in ion flux may be assessed by determining changes inpolarization (i.e., electrical potential) of the cell or membraneexpressing the Kir channels of this invention. A preferred means todetermine changes in cellular polarization is by measuring changes incurrent (thereby measuring changes in polarization) with voltage-clampand patch-clamp techniques, e.g., the “outside-out” mode, and the “wholecell” mode (see, e.g., Ackerman et al., New Engl. J. Med. 336:1575-1595(1997) and Single Channel Recording, Plenum Press, B. Sakmann and E.Neher eds). Whole cell currents are conveniently determined using thestandard methodology (see, e.g., Hamil et al., P Fingers. Archly. 391:85(1981). Other known assays include: radiolabeled rubidium flux assaysand fluorescence assays using voltage-sensitive dyes (see, e.g.,Vestergarrd-Bogind et al., J. Membrane Biol. 88:67-75 (1988); Daniel etal., J. Pharmacol. Meth. 25:185-193 (1991); Holevinsky et al., J.Membrane Biology 137:59-70 (1994)). Assays for compounds capable ofinhibiting or increasing potassium flux through the channel proteins canbe performed by application of the compounds to a bath solution incontact with and comprising cells having an channel of the presentinvention (see e.g., Blatz et al., Nature 323:718-720 (1986); Park, J.Physiol. 481:555-570 (1994)). Generally, the compounds to be tested arepresent in the range from 1 pM to 100 μM.

The effects of the test compounds upon the function of the Kir channelscan be measured by changes in the electrical currents or ionic flux orby the consequences of changes in currents and flux. Changes inelectrical current or ionic flux are measured by either increases ordecreases in flux of cations such as potassium or rubidium ions. Thecations can be measured in a variety of standard ways. They can bemeasured directly by concentration changes of the ions or indirectly bymembrane potential or by radiolabeling of the ions. Consequences of thetest compound on ion flux can be quite varied. Accordingly, any suitablephysiological change can be used to assess the influence of a testcompound on the Kir channels of this invention. The effects of a testcompound can be measured by a toxin binding assay. When the functionalconsequences are determined using intact cells or animals, one can alsomeasure a variety of effects such as transmitter release (e.g.,dopamine), hormone release (e.g., insulin), transcriptional changes toboth known and uncharacterized genetic markers (e.g., northern blots),cell volume changes (e.g., in red blood cells), immunoresponses (e.g., Tcell activation), changes in cell metabolism such as cell growth or pHchanges, and changes in intracellular second messengers such as [Ca²⁺].

Two Electrode Voltage Clamp Assay

One assay that may be of particular use in the present invention is atwo electrode voltage clamp assay. This assay may be performed using anyof these Kir channel proteins and compounds of the present inventionusing modifications readily known to those in the art. In the examplebelow, this assay was conducted using the chicken Kir 2.2 channel. Toperform this assay, Xenopus oocytes will be harvested from mature femaleXenopus laevis and defolliculated by collagenase treatment for 1-2hours. Oocytes will then rinsed thoroughly and stored in ND96 solution(96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1.0 mM MgCl₂, 5 mM HEPES, 50 μg/mlgentamycin, pH 7.6 with NaOH). Defolliculated oocytes will be selected2-4 hours after collagenase treatment and injected with cRNA the nextday. The injected oocytes will be incubated in ND96 solution for 1-5days before recording. All oocytes will be stored in an incubator at 18°C.

The desired human or chimeric Kir channel protein DNA will be sub-clonedinto the pGEM vector (Promega). cRNA will be prepared using T7 RNApolymerase (Promega) from NdeI-linearized plasmid DNA.

All recordings will be performed at room temperature. For two-electrodevoltage-clamp experiments, oocytes will be held at 0 mV and pulsed from−80 mV to +80 mV with 10 mV increment steps. Recording solution willcontain 98 mM KCl, 0.3 mM CaCl₂, 1 mM MgCl₂, and 5 mM HEPES pH 7.6. Theionic currents will be recorded with an oocyte clamp amplifier (OC-725C,Warner Instrument Corp.). The recorded signal will be filtered at 1 kHzand sampled at 10 kHz using an analogue-to-digital converter (Digidata1440A, Axon Instruments, Inc) interfaced with a computer. pClamp10.1software (Axon Instruments, Inc) will be used for controlling theamplifier and data acquisition.

Patch Clamp Assays

Patch clamp assays use a micropipette attached to a cell membrane toallow recording from a single ion channel in the cell membrane.

To perform this type of assay a micropipette which serves as amicroelectrode is positioned next to a cell, and a piece of the cellmembrane (the ‘patch’) is drawn into the microelectrode tip; the glasstip of the micropipette forms a high resistance ‘seal’ with the cellmembrane, then whole cell mode is entered by applying suction. Next, thepipette is moved away from the cell to form an outside-out patch.Examples of useful protocols may be found in Single Channel Recording,Plenum Press, B. Sakmann and E. Neher eds. This configuration can beused to study Kir channels present in the isolated patch of membrane.Variations of this technique include the “perforated patch” technique,or the patch of membrane can be pulled away from the rest of the cell.

As an example, for patch-clamp experiments in the outside-out mode, eachoocyte will be incubated in a hypertonic solution containing 200 mMNaCl, 130 mM KCl, 5 mM K₂EDTA, 5 mM K₂HPO₄, 5 mM KH₂PO₄ pH 7.2 for 5-10minutes and the vitelline membrane will be removed before sealformation. Currents will be recorded in either cell-attached oroutside-out configuration with an Axopatch 200B amplifier, Digidata1440A analogue-to-digital converter and pClamp10.1 software to controlmembrane voltage and record. During the current recordings, the membranewill be first held at 0 mV followed by a 10-second voltage ramp from +80mV to −80 mV. The pipette solution will contain 140 mM KCl, 5 mM K₂HPO₄,5 mM KH₂PO₄, 0.3 mM CaCl₂, 1 mM MgCl₂, pH 7.2 with KOH. The bathsolution will contain 130 mM KCl, 5 mM K₂EDTA, 5 mM K₂HPO₄, 5 mM KH₂PO₄,pH 7.2 with KOH.

To measure a compound for activity, first a control current is measuredwhile perfusing with the recording solution without the compoundpresent. Then, a second current is recorded while perfusing with thesolution and the compound of interest. Any difference in current levelsindicates that the compound acts to modulate the activity of the Kirchannel. An example of this type of assay may be found in Lu andMacKinnon 1997 Biochemistry, vol. 36, no. 23, pp. 6936-6940 or Namba etal., 1996 FEBS Letters vol. 386, pp. 211-214.

Planar Lipid Assay

Another electrophysiological assay which may be used in the presentinvention is a planar lipid bilayer assay. In this type of assay a lipidbilayer is created and the Kir channel protein is introduced into thelipid bilayer. A hydrophobic material such as Teflon is used to preparethe lipid bilayer by making a small hole (an aperture) in a sheet ofTeflon. A syringe containing a solution of lipids dissolved in anorganic solvent is introduced to the hole and a bilayer is formed in thecenter of the aperture, with solvent forming the perimeter of the newlyformed bilayer.

The Teflon sheet provides a partition between two chambers allowing theplacement of electrodes on both sides of the sheet. Preferably, thepurified Kir channel is reconstituted into lipid vesicles and then fusedwith the bilayer after it is formed. The detergent coating facilitatesinsertion into the bilayer. (See U.S. Pat. No. 6,191,254 and Guillermoet al J. Membrane Biol (2008) 223: 13-26).

The amount of lipid desired (preferably PE:PG 3:1) is pipetted into aglass vial (about 5-10 mg). The lipid is dried under argon and thenfurther under a room temperature vacuum for about 3 hours.

The lipid is rehydrated with hydration buffer (10 mM HEPES 7.4 (KOH),450 mM KCl, 4 mM N-methylglucamine, 2 mM DM to a final lipidconcentration of 10 mg/ml and vortexed briefly. The glass vial isflushed with argon and the lipid mixture is sonicated mildly, i.e., withshort pulses of no longer than 30 seconds each. In between sonicationpulses, the lipid mixture is cooled in a room temperature water bath toensure that the lipid mixture does not get too hot. This procedure isrepeated until the lipid mixture becomes translucent with a distinctpink shade.

A solution containing 50 mM DM in the hydration buffer is prepared. TheDM solution is added to the lipid mixture to give a final concentrationof 10 mM of DM and rotated at room temperature for 2 hours. To thedetergent/lipid mixture, the Kir channel is added to the desired ratios(e.g., about 0.05-0.1). The concentration of DM is then raised to 17.5mM and the mixture is rotated at room temperature for 1 hour. Thedetergent/lipid mixture is then put into dialysis tubing and dialysedagainst the hydration buffer.

Fluorescent Dye Assay

Another assay which may be used to identify compounds which specificallybind the Kir channel turret region is an assay utilizing a fluorescentdye. An example of a suitable dye is FIuxOR™ available from Invitrogen(catalog nos, F10016, E10017), An example of this method is shown inFLOWCHART 2.

The FluxOR™ reagent is a fluorogenic indicator dye, which is loaded intocells as a mernbrance-permeable Acetoxymethanol (AM) ester. According tothe protocol, the FluxOR™ reagent is dissolved in DMSO and furtherdiluted with the FluxOR™ assay buffer, a physiological Hank's balancedsalt solution, for loading into cells. Pluronic® surfactants, whichdisperse and stabilize the dye are used to facilitate loading in aqueoussolution.

Mammalian cells such as HEK, COS or CHO cells are grown in culture andincubated with the dye. Inside the cell, the non-fluorescent AM esterform of the FluxOR™ dye is cleaved by endogenous esterases into aflourogenic thallium-sensitive indicator. The thallium-sensitive form isretained in the cytosol and its extrusion is inhibited by water-solubleProbenecid, which blocks organic anion pumps. The dye-loading buffer isreplaced with fresh, dye-free assay buffer, composed of physiologicalHBSS containing Probenecid, before the assay. During the assay, a smallamount of thallium is added to the cells with a stimulus solution thatopens potassium-permeant ion channels with a mild depolarization oragonist addition. Thallium then passes into cells through open potassiumchannels according to a strong inward driving force. Upon bindingcytosolic thallium, the de-esterified FluxOR™ dye exhibits a strongincrease in fluorescence intensity at its peak emission of 525 mmBaseline and stimulated fluorescence is monitored in real time to give adynamic, functional readout of thallium redistribution across themembrance with no interference from quencher dyes.

inhibitors such as, for example, the compounds of the present inventionmay slow the rate of entry of thallium and thus reduce the onset of afluorescent signal. This assay may be used for the selection ofcompounds that specifically bind to the turret regions of the Kirproteins. To identify such compounds a first group of cells would betransfected with wild type Kir channels and a second group of cellstransfected with Kir channel having mutated turrets as illustrated inFLOWCHART 2. Test compounds such as the antibodies identified in theassays above would be added to the cells to screen for those compoundswhich inhibit or reduce the onset of fluorescence upon addition of thethallium dye due to inhibition of the channel. Compounds which reducedthe rate of thallium intake in cells with normal turrets but had noeffect on cells with mutant turrets would be classified as turretspecific inhibitor compounds.

This assay may also be used to determine the specificity of thecompounds for given turrets, in other words, the compounds may beintroduced into cells which have been transfected with differentversions of the Kir Channel to determine if the compound is specific fora given type of Kir Channel protein.

Assay for Selective Binding to Specific Types of Kir Channels

In order to determine whether a given antibody is specific for a giventype of Kir Channel, assays such as an ELISA assay may be performed inwhich an antibody is tested against a variety of different Kir Channelsto determine if the antibody is specific for a single type of KirChannel. Ideally, antibodies that would be used as therapeutic compoundswill bind to only one type of Kir channel in the turret region.

Methods to Identify Compounds to Treat Conditions

Compounds that bind to the turret region of a Kir channel and whichmodulate the ion channel activity of a Kir channel may be administeredto a subject to determine if such compounds are able to treat a givencondition. As an example, a compound may be administered to a subjectsuch as a mammal with a given disease state using known methods ofadministration and the subject is then monitored clinically and testedusing biochemical assays to determine if the compound is able to treatthe condition using known assays for the disease state. It is believedthat a variety of conditions may be treated with the compounds of thepresent invention, including, but not limited to, diabetes mellitus,hypertension, cardiac arrhythmia and epilepsy.

The present invention may be better understood by reference to thefollowing non-limiting example, which is provided as exemplary of theinvention. This example should in no way be construed, however, aslimiting the broad scope of the invention. Example 1, which followsbelow, provides the first determination of the crystal structure of aeuraryotic Kir channel protein and identification of the structuredturret region present in Kir channel proteins. (See Crystal Structure ofthe Eukaryotic Strong Inward-Rectifier K ⁺ Channel Kir2.2 at 3.1 ÅResolution, X. Tao, J. L. Avalos, J. Chen and R. MacKinnon, Science 2009December 18; 326 (5960); 1668.)

Example 1 Crystal Structure of the Eukaryotic Strong Inward-Rectifier K⁺Channel Kir2.2 at 3.1 Å Resolution

Inward-rectifier K⁺ channels conduct K⁺ ions most efficiently in onedirection, into the cell. Kir2 channels control the resting membranevoltage in many electrically excitable cells and heritable mutationscause periodic paralysis and cardiac arrhythmia. We present the crystalstructure of Kir2.2 from chicken, which, excluding the unstructured N-and C-termini, is 90% identical to human Kir2.2, Crystals containingRb⁺, Sr²⁺ and Eu³⁺ reveal binding sites along the ion conduction pathwaythat are both conductive and inhibitory. The sites correlate withextensive electrophysiological data and provide a structural basis forunderstanding rectification. The channel's extracellular surface, withlarge structured turrets and an unusual selectivity filter entryway,might explain the relative insensitivity of eukaryotic inward rectifiersto toxins. These same surface features also suggest a possible approachto the development of inhibitory agents specific to each member of theinward-rectifier K⁺ channel family. A crystal structure reveals thestructural basis of diode-like conduction properties and relative toxininsensitivity in inward rectifier K⁺ channels.

Introduction

In 1949 Bernard Katz introduced the term ‘anomalous rectification’ todistinguish the K⁺ currents he observed in frog skeletal muscle from the‘delayed rectification’ K⁺ currents of the squid axon action potential(1, 2). Today we know that ‘delayed rectifiers’ are a subset of thelarge family of voltage-dependent K⁺ (Kv) channels, while ‘anomalousrectifiers’ are members of a different family of channels more commonlyknown as inward rectifier K⁺ (Kir) channels (3). The name inwardrectifier refers to a fundamental ion conduction property exhibited to agreater or lesser degree by all members of the family: given an equalbut opposite electrochemical driving force, K⁺ conductance into the cellfar exceeds conductance out of the cell. Thus, Kir channels areanalogous to one-way conductors, or diodes, in solid-state electronicdevices.

Electrophysiological experiments have shown that inward rectification isa consequence of voltage-dependent pore blockage by intracellularmultivalent cations, especially Mg²⁺ and polyamines (4-8). At internalnegative (hyperpolarizing) membrane voltages the blocking ions arecleared from the pore so that K⁺conducts, whereas at internal positive(depolarizing) membrane voltages the blocking ions are driven into thepore from the cytoplasm so that K⁺ conduction is blocked. As a result,Kir channels are conductive when an excitable cell is at rest andnon-conductive during excitation. This property is thought to fosterenergy efficiency because it enables Kir channels to regulate theresting membrane potential, but not dissipate the K⁺ gradient during anaction potential (3).

A central mechanistic question is why are Kir channels blocked byintracellular multivalent cations? Mutational studies have identifiedseveral amino acids that confer sensitivity to blocking ions (9-19), buta structural description of these sites has remained elusive. Structuresof prokaryotic Kir channels, due to their low sequence similarity toeukaryotic Kir channels, do not contain the specific amino acids thatare known to underlie blockage and rectification (20, 21).

Another longstanding puzzle in eukaryotic Kir channel studies is theirrelative insensitivity to natural toxins that typically inhibit other K⁺channels (22-24). Snake, spider and scorpion venoms, for example,contain numerous toxins against various Kv channels and Ca²⁺-activatedK⁺ channels (25-27). By contrast, Kir channel toxins are rare, and nospecific toxins against Kir2 channels have been discovered.

Results and Discussion Eukaryotic Kir Channels as a Molecular Family

The eukaryotic Kir channels contain several amino acid sequence motifsand conserved amino acids that are essential to their functionalproperties (FIGS. 1A-1C). For example, in most other K⁺ channels theselectivity filter comprises the ‘canonical’ filter sequence TXGYGDX,where X represents an aliphatic amino acid (FIGS. 1A-1C). Thecorresponding sequence in eukaryotic Kir channels is TXGYGFR, with Fsometimes replaced by another amino acid. In light of the structuralimportance of DX in the canonical sequence, the amino acids FR signify amarked variation on the filter sequence. Eukaryotic Kir channels alsocontain an absolutely conserved pair of cysteine residues flanking thepore-region, which is the re-entrant peptide segment that forms thepore-helix and selectivity filter of K⁺ channels. Between the outerhelix (the first transmembrane segment) and pore-region the ‘turret’,though varied amongst inward rectifiers, contains the sequence HGDL thatcould be considered a signature of eukaryotic Kir channels, Finally,through extensive studies combining electrophysiology and mutagenesisseveral acidic amino acids (D and E) are known to be critical to inwardrectification (9-19), and motifs containing basic amino acids (e.g.PKKR) are critical to PIP₂ activation of Kir channels (28-35). Thesepositions are enclosed in boxes on the sequences in FIGS. 1A-1C.

The Kir2.2 channel from chicken is 90% identical to the human ortholog(excluding the N- and C-termini) and contains all of the sequencecharacteristics of a strong inward rectifier (36). FIGS. 6A-6D showsthat the chicken Kir2.2 channel expressed in Xenopus oocytes indeedfunctions as a strong rectifier. In oocyte two-electrode voltage clamprecordings with 98 mM KCl in the bath solution inward currents are muchlarger than outward currents (FIG. 6B). In on-cell and excised gigasealpatch recordings channel activity is observed at hyperpolarizing(negative internal) membrane voltages but not at depolarizing (positiveinternal) voltages (FIG. 6C). The single channel conductance measurednear −80 mV is approximately 40 pS, which is very similar to the valuesreported for the guinea pig and mouse Kir2.2 channels (37, 38) (FIG. 6D,inset). The sharp transition between channel conductance andnon-conductance as a function of membrane voltage is characteristic of astrong rectifier (36). Note that upon patch excision from the oocytesurface some outward current is observed at voltages slightly positiveto the reversal potential because the concentration of intracellularblockers is decreased (FIG. 6C, trace labeled (C)). However, the currentstill decreases with further depolarization (negative conductance) aschannels become blocked in a voltage-dependent manner: this behaviorreflects the inherent difficulty in washing away trace yet still activeconcentrations of polyamine molecules due to their very high affinityfor the pore in strong rectifiers (39, 40). Several minutes followingpatch excision the currents decrease (FIG. 6C, trace labeled (A)). This‘run-down’ reflects altered channel regulation mediated by kinases,phosphatases and lipid signaling (34, 36, 41, 42).

In order to obtain diffracting crystals the intrinsically disordered N-and C-terminal regions were removed, The electrophysiological recordingsshown in FIGS. 6A-6D were made using a similar construct with N- andC-terminal truncations, confirming that the crystal structurecorresponds to a functional channel unit with strong rectifyingproperties. The Kir2.2 model, consisting of the cytoplasmic domain andtransmembrane channel, was refined at 3.1 Å to a free R-factor of 0.27.A ribbon diagram in stereo shows the transmembrane pore (above) and thecytoplasmic pore (below) (FIG. 2A). Lateral openings between thetransmembrane and cytoplasmic pores, at the level of the lipid membraneheadgroup layer, contain many arginine and lysine residues. The highdensity of positive charges makes it unlikely that K⁺ ions would passthrough these openings (FIG. 7). In FIG. 7 the shading at the top of theFigure illustrates a negative electrostatic potential at the surface andthe darker shaded region in the center of the Figure illustrates regionsof positive electrostatic potential. The structure is thereforeconsistent with mutagenesis studies, which support the conclusion thatthe ion pathway extends across the full length of the transmembrane andcytoplasmic pores (9-19). The overall architecture is similar toprokaryotic Kir channels but with a notable difference: the Kir2.2channel contains prominent, highly structured turrets on theextracellular face of the channel. These surround as if to protect thepore entryway.

The Selectivity Filter

At a detailed structural level Kir2.2 is quite different fromprokaryotic Kir channels owing to minimal (<20%) sequence conservation.The cysteine pair that is absolutely conserved among eukaryotic Kirchannels creates a circularized pore region through covalent linkage ofthe segment preceding the pore helix (C123) to the segment following theselectivity filter (C155) (FIG. 2B). The existence of a disulfide bondwas correctly predicted on the basis of mutagenesis studies: mutation ofthe corresponding cysteines in Kir2.1 led to the absence of currentseven though expressed protein was detectable by Western Blot analysis(43, 44). Application of 10 mM DTT or reduced glutathione to the outsideof cells expressing the wild-type channels did not affect currents. Fromthese two observations it was concluded that a disulfide bridge must beessential for proper folding, but apparently not for function (43, 44),The structure provides an alternative interpretation. The disulfidebridge is buried beneath the protein surface at the level of themembrane interface. Furthermore, the Kir2.2 channel was purified andcrystallized in the presence of 20 mM DTT and 3 mM TCEP, and yet thedisulfide bridge remained intact. It is therefore possible that thedisulfide bridge remains intact upon exposure to moderate concentrationsof DTT, and that the bridge may be important for channel function.

The pore region is further stapled together by an ionized hydrogen bondbetween R149 in the filter sequence TXGYGFR and E139 (FIGS. 2B and 2C).The Glu O-ε to Arg distance is 2.4 Å, compatible with an energeticallystrong interaction. Mutations altering this interaction are known toalter channel function (45, 46). On the basis of studies withconcatenated subunits the salt bridge was thought to be inter-subunit,but the crystal structure shows that this interaction ties together twosegments of the pore-region within a single subunit (46).

Despite the presence of substantially different protein contactssurrounding the selectivity filter, the main-chain structure of thefilter in Kir2.2 is the same as in other K⁺ channels (47). For example,the main chain RMSD between Kv1.2 and Kir2.2 is 0.4 Å, which is withinthe margin of certainty to discriminate atomic positions with 3.1 Ådiffraction data (FIGS. 2D and 2E) (20, 48, 49), One structuraldifference near the filter could possibly account for importantpharmacological differences between Kir and other K⁺ channels. In thecanonical filter sequence the Asp (D) residue in the filter sequence isburied, creating a flat surface surrounding the filter opening. Bycontrast, in Kir channels the Phe (F) residue at the correspondingposition projects directly into aqueous solution, creating fourprotrusions on the perimeter where the filter opens to the extracellularsolution.

The Cavity and Gates

The pore lining on the intracellular side of the selectivity filter ismainly hydrophobic in nearly all K⁺ channels. Eukaryotic Kir channelsare an exception in which the central region of the pore—known as thecentral cavity—contains four polar amino acids (one from each subunit)projecting toward the ion pathway (FIG. 3A). In Kir2.2 and other strongrectifiers these polar amino acids are Asp (D173), whereas in weakrectifiers such as Kir1.1 and Kir6.1 they are Asn (FIG. 1A-1C). On thebasis of electrophysiological studies, Asp residues in the centralcavity of strong rectifiers are hypothesized to influence the affinityof Mg²⁺ and polyamines by an electrostatic mechanism (12, 18).

Beneath the central cavity, residues I177 and M181 on the inner helicesform two hydrophobic seals that close off the pore leading to thecytoplasm (FIG. 3C). Kir2.2 is therefore physically shut at the‘activation gate’ (50). Amino acids corresponding to positions 177 and181 are also large and hydrophobic in most other eukaryotic Kirchannels, but not in many other K⁺ channels (FIGS. 1A-1C). For example,in KcsA, Kv channels and prokaryotic Kir channels, the positioncorresponding to 177 usually contains a small and sometimes polar aminoacid, typically Ala or Thr. In KcsA both seal positions contain smallamino acids (FIG. 3D). Because of the large hydrophobic residues atpositions 177 and 181, the inner helices of Kir2.2 do not come as closetogether in the closed conformation as in KcsA (FIGS. 3C and 3D).

FIG. 3E shows the cytoplasmic domain tetramer from the Kir2.2 channelsuperimposed onto the domain from Kir2.1, which was solved bycrystallography in the absence of a transmembrane channel (11). Overmost of the domain these structures are nearly identical. Thisobservation supports the expectation (based on 80% sequence identity)that Kir2.2 should represent an excellent model for the complete Kir2.1channel. In addition to the activation gate formed by the transmembraneinner helices, Kir channels have been proposed to have a second gate(G-loop) at the apex of the cytoplasmic domain tetramer (11, 51). TheG-loop is physically open in Kir2.2 and closed in the Kir2.1 domain(FIGS. 3F and 3G). The differences in conformation are due to localmovements of the G-loop rather than rigid body motions of thecytoplasmic domains. Local G-loop movements contrast observations on thecytoplasmic domain of Kir3.1, in which G-loop opening appears associatedwith rigid body movements of domains in the tetramer (20).

Ion Binding Sites for Conduction and Inward Rectification

FIG. 4A-F shows the locations of ions in difference Fourier maps fromcrystals containing Rb⁺, Sr²⁺, and Eu³⁺. Rb⁺ is a K⁺ analog thatconducts. Density for this ion is observed at multiple sites in theselectivity filter and at three positions within the pore on theintracellular side of the selectivity filter, but is absent in thecentral cavity (FIG. 4A). The three occupied intracellular positionsare: immediately internal to the activation gate in the transmembranepore, in the cytoplasmic pore internal to the G-loop, and at theentryway to the cytoplasmic pore. We refer to the two sites in thecytoplasmic pore as the upper and lower rings of charges, respectively(FIG. 8). The presence of multiple sites along the pore occupied byconducting ions area prerequisite for strong voltage-dependent block byintracellular cations that cannot pass through the selectivity filter(12, 52-57).

Crystals of Kir2.2 were grown in the presence of 650 mM Rb⁺ and yetelectron density for Rh⁺ is not observed in the cavity (FIG. 4A). Thisfinding is noteworthy because under similar conditions a strongmonovalent cation peak is observed in the cavity of KcsA (47, 58).Native crystals of Kir2.2, grown in the presence of 150 mM K⁺ and 500 mMNa⁺, show a weak electron density peak at the cavity center withadditional peaks on the perimeter, apparently bridging toward the D173side-chain (FIG. 3A). We can not discern whether these peaks represent adisordered ion, multiple ions, or a low occupancy K⁺ (or Na⁺) in thecenter, perhaps surrounded by water molecules hydrogen bonded to the Aspcarboxylate. We can conclude, however, that the central cavity inKir2.2, at least in the closed conformation, has cation attractiveproperties that are different from KcsA.

The divalent cation Sr²⁺ should behave as an electron dense mimic ofMg²⁺, a biologically important metal ion inhibitor of eukaryotic Kirchannels (7, 8). In F_(o)-F_(c) Fourier maps from crystals with 10 mMSi²⁺, 500 mM Na⁺ and 150 mM K⁺, density peaks due to Sr²⁺ are observedat three sites inside the pore intracellular to the selectivity filter:in the cavity, at the upper ring and at the lower ring of charges (FIGS.8 and 4B). The magnitude of the Sr²⁺ peak is small in the cavity (3.4σ)compared to the peaks at the upper (9.6σ) and lower (7.2σ) rings ofcharges. Separate experiments with crystals containing 200 mM Sr²⁺support that the weak cavity peak is indeed due to Sr²⁺, which ispresent apparently at relatively low occupancy. Detailed views of thesesites are shown (FIGS. 4D-F). They each consist of planar rings ofacidic amino acids arranged on the pore's perimeter. All three sitesexhibit a preference for Sr²⁺:10 mM Sr²⁺ out competes 150 mM K⁺. Thisselectivity is likely to be electrostatic in origin. The sites are toowide (10.5 Å, 8.9 Å and 9.3 Å diameter for the cavity, upper and lowerring of charges) to mediate direct coordination of an ion at the center.Presumably ions at the center of these sites interact through bridgingwater molecules. Since each site has the potential to contain multiplenegatively charged carboxyl groups, the resulting strong electric fieldis expected to create a good match for a multivalent cation. Crystalscontaining the lanthanide Eu³⁺, which we assume to be trivalent (59),provide support for this hypothesis. An anomalous difference Fourier mapshows that Eu³⁺ binds at only one site, the upper ring of charges. Thissite appears to be more electronegative than the others because itcontains two concentric rings of acidic amino acids, E225 and E300.

Mutagenesis studies have identified several amino acids that, whenmutated, affect the affinity of Mg²⁺ and polyamines in strongrectifiers. D173 in the cavity, E225 and E300 forming the upper ring ofcharges, and D256 forming the lower ring of charges are among thoseknown to be important (9-19). The weak Sr²⁺ peak in the cavity mightseem incompatible with the large influence that mutations of the cavityAsp (D173) have on Mg²⁺ affinity. However, the channel in the crystal isnot in an applied electric field: in an electric field imposed by adepolarized (positive inside) membrane we expect that the distributionof blocker occupancies among the multiple sites will change.Specifically, we expect the blocking cations to be driven deeper intothe pore toward the cavity. In correlating the crystallographic withelectrophysiological data, it is most significant that the amino acidsforming the Sr²⁺ sites in the crystal are the same amino acids that areknown to affect blockage and rectification in electrophysiologyexperiments (36). Beyond providing a structural basis with which toexplain past electrophysiological studies, the Kir2.2 structure alsosuggests many new experiments. For example, most studies on themechanism of rectification have focused on electrostatic interactionsbetween the positively charged blocker and negatively charged groups onthe protein. But hydrophobic interactions between methylene groups ofpolyamine molecules and hydrophobic residues in the channel may beimportant. In particular, we might anticipate that when the pore openspolyamines could interact strongly with the large hydrophobic aminoacids at positions 177 and 181 when the leading amino group of thepolyamine reaches into the central cavity (FIG. 3C) (54).

Since the earliest investigations of strong inward rectifiers twoimportant properties have been noted: a sharp transition from aconductive state to a non-conductive (blocked) state over a very narrowvoltage range, and a dependence of the transition on the extracellularK⁺ concentration (60-63). Specifically, the voltage at which thetransition occurs shifts to more depolarizing values as extracellular K⁴concentration is increased. Both properties, the sharp transition (i.e.strong voltage dependence) and its dependence on extracellular K⁺, havebeen attributed to the simple notion that conducting ions and blockingions compete for sites in the pore (12, 52-57, 64-66). Thecrystallographic data presented here support this conclusion. We observein the crystal Rb⁺binding at the same sites that can bind multivalentblocking ions. Therefore a high extracellular K⁺ (or Rb⁺) concentrationshould favor occupation of the sites by conducting ions, and a moredepolarizing voltage should be required to drive blocking ions into thepore from the cytoplasm to replace the conducting ions. Moreover, asblocking ions enter the pore from the intracellular side, the displacedconducting ions must move through the selectivity filter to theextracellular side. This is to say that movements of blocking andconducting ions must be coupled. Such coupling would have energeticconsequences because movement of an ion across the membrane voltagedifference constitutes work. In other words a blocking ion entering thepore will exhibit a voltage dependence that results from a combinationof its own charge and the charge of the displaced ions. This can be theorigin of strong voltage dependent block, which can be the origin of abiologically important property of strong rectifiers—their diodeproperty of a sharp transition from a conductive to a non-conductivestate as a function of membrane voltage (12, 52-55, 64).

The Extracellular Pore Entryway and Pharmacology of Kir Channels

Two aspects of the structure may account for the fact that eukaryoticKir channels, especially members of the Kir2 subfamily, are relativelyinsensitive to K⁺ channel toxins (22-24). The turrets in Kir2.2 arelarger and come closer together, constricting the pore entryway comparedto Kv1.2; and F148 in the sequence TXGYGFR creates four protrusions onthe surface at the pore opening (FIGS. 5A and 5B). Thus, in Kv channelsthe entryway is wider and the pore opens onto intersecting grooves witha flat base, which form the docking surface for pore-blocking scorpiontoxins (FIG. 5B). In Kir2.2 the entryway is constricted and the groovesare absent (FIG. 5A).

Though the shape of the eukaryotic Kir channel pore entryway might offerfewer opportunities for inhibitory protein-protein interactions,inhibition might occur by a somewhat different strategy. Inhibitors ofKir1.1 and Kir3.4 channels have been identified. A bee venom toxin,tertiapin, inhibits both of these channels (22). At 21 amino acidstertiapin is smaller than most other venom toxins so it might fitbetween the turrets more effectively, Alternatively, the turretsthemselves might form the binding site for tertiapin (67-69). At 57amino acids δ-dendrotoxin from the green mamba snake is rather large andyet it inhibits Kir1.1 channels (23). Compared to tertiapin less isknown about the binding site on the channel for δ-dendrotoxin, but oneaspect of its inhibition is intriguing: the blocked state reduces singlechannel conductance to about 10% rather than inhibiting all the way.δ-dendrotoxin most likely binds to the turrets but is too large to fittightly over the pore, which would imply that binding to the turret maybe sufficient to alter the channel's function.

The idea that binding to the turrets could alter function is notsurprising when one considers that the turret in Kir2.2 is not a loop,but forms a highly ordered structure (FIG. 5C). The base of the turretis formed and pinned together by the HGDL sequence, which with onlyminor variation is found in all eukaryotic Kir channels (FIGS. 1A-1C andFIG. 5D.) H108 stabilizes D110 through a hydrogen bond. The Asp (D)itself is hydrogen bonded to the amide nitrogen of C123, whicheffectively holds the two ends of the turret together. L111 projectsfrom the surface of a short 3₁₀ helix into the protein interior to makestabilizing hydrophobic interactions. Thus, the turrets are structurallyimportant elements of the channel. Between the sequence HGDL and thefirst Cys of the disulfide bridge the turret sequence is highly variableamong Kir channel subtypes. The Kir2.1 channel becomes sensitive totertiapin if the variable sequence is mutated to be Kir3.4-like (68).Therefore, the turrets appear to be structures through which specificinhibition of Kir channel subtypes might be achievable through directedevolution of specific protein binding partners.

SUMMARY

This presents the atomic structure of a eukaryotic Kir channel, Kir2.2,a strong inward rectifier. The sequence TXGYGFR gives rise to a K⁺selectivity filter stabilized by disulfide bridges and salt bridges thatdistinguish eukaryotic Kir channels. Multiple ion binding sites on theintracellular side of the selectivity filter can be occupied byconducting ions but exhibit higher affinity for multivalent blockingions. Thus, blocking ions entering from the cytoplasm must displaceconducting ions through the pore. This situation is expected to giverise to strong voltage-dependent block and diode-like conductionproperties. Structural features of the extracellular pore entryway offeran explanation for the relative insensitivity of Kir channels tovenomous toxins and a possible approach to the development of selectiveKir channel inhibitors,

REFERENCES AND NOTES

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Materials and Methods Cloning, Expression and Purification

A synthetic gene fragment (Bio Basic, Inc) encoding residues 38 to 369of chicken Kir2.2 channel (GI:118097849) was ligated into the XhoI/EcoRIcloning sites of a modified pPICZ-B vector (Invitrogen). The resultingprotein has green fluorescent protein (GFP) and a 1D4 antibodyrecognition sequence (TETSQVAPA) on the C-terminus (1), separated by aPreScission protease cleavage site (SNSLEVLPQ/GP).

The construct was linearized using PmeI and transformed into a HIS⁺strain of SMD1163 of Pichia pastoris (Invitrogen) by electroporation(BioRad Micropulser). Transformants were selected on YPDS platescontaining 400-1200 μg/ml Zeocin (Invitrogen). Resistant colonies weretested for expression by anti-1D4 tag Western Blot. For large-scaleexpression, small cultures grown from the best expressing colony werediluted into BMGY media (Invitrogen) and inoculated at 29° C. overnight,until OD₆₀₀ reached between 20-30. Cells were then pelleted, resuspendedin BMM media (Invitrogen) and expressed overnight at 24° C. Cells wereharvested, flash-frozen in liquid N₂, and stored at −80° C. untilneeded.

Cells were lysed in a Retsch, Inc. Model MM301 mixer mill (5×3.0 minutesat 25 cps). The lysis buffer contained 150 mM KCl, 50 mM TRIS-HCl pH8.0, 0.1 mg/ml deoxyribonuclease I, 0.1 μg/ml pepstatin, 1 μl/mlleupeptin, 1 μg/ml aprotinin, 0.1 mg/ml soy trypsin inhibitor, 1 mMbenzamidine, 0.1 mg/ml AEBSF, with 1 mM phenylmethysulfonyl fluorideadded just before lysis (3.0 ml lysis buffer/g cells). pH of the lysatewas adjusted to 8.0 with KOH. The lysate was extracted with 100 mM DM(n-decyl-β-D-maltopyranoside, Anatrace, solgrade) at room temperaturefor 1 hour with stirring, and then centrifuged for 40 minutes at 30,000g, 10° C. Supernatant was added to 1D4-affinity resin pre-equilibratedwith 150 mM KCl, 50 mM TRIS-HCl pH 8.0, and 4 mM DM. Suspension waslayered with Argon and mixed by inversion for 2 hours at roomtemperature. Beads were collected on a column by gravity, washed with 2column volumes of buffer (150 mM KCl, 50 mM TRIS-HCl pH 8.0, 1 mM EDTApH 8.0, and 4 mM DM), and eluted with buffer plus 1 mg/ml 1D4 peptide(AnaSpec, Inc.) over 1 hour at room temperature. 20 mM DTT(Dithiothreitol) and 3 mM TECP were added to eluted protein. The proteinwas then digested with PreScission protease (20:1 w/w ratio) overnightat 4° C. Concentrated protein was further purified on a Superdex-200 gelfiltration column in 150 mM KCl, 20 mM TRIS-HCl pH 8.0, 4 mM DM(anagrade), 3 mM TCEP, 20 mM DTT and 1 mM EDTA at 4° C.

The fraction corresponding to the tetramer peak was concentrated toabout 8 mg/ml, mixed 1:1 with crystallization solution and set up ashanging drops over reservoirs containing 0.1 ml crystallizationsolution. Crystals appeared in 7-20% PEG400 or 2-10% PEG4000, with 500mM KCl or NaCl, and 50 mM buffer pH 6.0-9.5 at 4° C. overnight and grewto full size within 2-3 days.

For studies with RbCl, the protein was purified in a similar fashionexcept that KCl was replaced with RbCl in all buffer solutions andcrystals were grown in 10-20% PEG400, 500 mM RbCl, and 50 mM MES pH 6.5.For studies with 10 mM EuCl₃, crystals were grown in 7-20% PEG400, 1 Mammonium formate, 50 mM TRIS-HCl pH 8.5, and 10 mM EuCl₃. For studieswith 10 mM SrCl₂, crystals were grown in 10-20% PEG400, 500 mM NaCl, 50mM HEPES pH 7.5, and 10 mM SrCl₂. For studies with 200 mM SrCl₂,crystals were grown in 3-7% PEG4000, 200 mM SrCl₂, and 50 mM Na CitratepH 5.6.

Structure Determination

Crystals were cryo-protected in reservoir plus 25% glycerol (v/v), 4 mMDM, 20 mM DTT, 3 mM TCEP, and 1 mM EDTA in a step-wise manner (5%glycerol increase each step) and flash-frozen in liquid nitrogen.Diffraction data from native crystals were collected to 3.1 Å atbeamline 24ID-C (APS) and for crystals in various metal ions (Rb⁺, Sr⁺,and Eu³⁺) at beamline X29 (Brookhaven NSLS). Images were processed withDENZO and intensities merged with SCALEPACK (2). Data were furtherprocessed using the CCP4 suite (3). The crystals belong to the 14 spacegroup. The structure was solved by molecular replacement using theprogram MOLREP (4), with the 2.4 Å resolution structure of thecytoplasmic domain of mouse Kir2.1 (PDB 1U4F) as a search model. Thereis one copy of the subunit in the asymmetric unit. The model was builtusing O (5) and refined with CNS (50-3.1 Å) to R_(free)=27.2% (6). Thefinal model contains residues 43-60 and 70-369 (residues 70 to 78 aremodeled as alanines) of chicken Kir2.2, three additional residues SNS onthe C-terminus corresponding to the PreScission cleavage site, and fiveK⁺ ions. During the final minimization refinement step in CNS,occupancies of the K⁺ ions were set to 0.5 (which gave rise to a lower Rfree compared to occupancy of 1.0) and B-factors of the K⁺ ions were setto 85 (roughly the average B-factor of surrounding protein atoms).Crystallographic data and refinement statistics are shown in Table S1.Figures were made using PYMOL (www.pymol.org) (7).

Ion binding was assessed by calculating anomalous difference Fouriermaps for data with Eu³⁺ and F_(o)-F_(c) maps for data with Rb⁺ and Sr²⁺using fft in the CCP4 suite (3), Sr²⁺ was analyzed at two differentconcentrations to discern whether the weak cavity peak was due to Sr²⁺.This peak became stronger when Sr²⁺ was increased from 10 mM to 200 mMwhile the monovalent cation concentration was decreased, consistent withSr²⁺ being present in the cavity but probably at low occupancy. Phasesused to calculate F_(o)-F_(c) omit maps were derived from a channelmodel devoid of ions in the cavity or cytoplasmic domain throughoutrefinement,

Electrophysiology

Xenopus oocytes were harvested from mature female Xenopus laevis anddefolliculated by collagenase treatment for 1-2 hours. Oocytes were thenrinsed thoroughly and stored in ND96 solution (96 mM NaCl, 2 mM KCl, 1.8mM CaCl₂, 1.0 mM MgCl₂, 5 mM HEPES, 50 μg/ml gentamycin, pH 7.6 withNaOH). Defolliculated oocytes were selected 2-4 hours after collagenasetreatment and injected with cRNA the next day. The injected oocytes wereincubated in ND96 solution for 1-5 days before recording. All oocyteswere stored in an incubator at 18° C.

The chicken Kir2.2 (residues 38 to 369) gene was sub-cloned into thepGEM vector (Promega). cRNA was prepared using T7 RNA polymerase(Promega) from NdeI-linearized plasmid DNA.

All recordings were performed at room temperature. For two-electrodevoltage-clamp experiments, oocytes were held at 0 mV and pulsed from −80mV to +80 mV with 10 mV increment steps. Recording solution contained 98mM KCl, 0.3 mM CaCl₂, 1 mM MgCl₂, and 5 mM HEPES pH 7.6. The ioniccurrents were recorded with an oocyte clamp amplifier (OC-725C, WarnerInstrument Corp.). The recorded signal was filtered at 1 kHz and sampledat 10 kHz using an analogue-to-digital converter (Digidata 1440A, AxonInstruments, Inc) interfaced with a computer. pClamp10.1 software (AxonInstruments, Inc) was used for controlling the amplifier and dataacquisition. For patch-clamp experiments, each oocyte was incubated in ahypertonic solution containing 200 mM NaCl, 130 mM KCl, 5 mM K₂EDTA, 5mM K₂HPO₄, 5 mM KH₂PO₄ pH 7.2 for 5-10 minutes and the vitellinemembrane was removed before seal formation. Currents were recorded ineither cell-attached or inside-out configuration with an Axopatch 200Bamplifier, Digidata 1440A analogue-to-digital converter and pClamp10.1software to control membrane voltage and record. During the currentrecordings, the membrane was first held at 0 mV followed by a 10-secondvoltage ramp from +80 mV to −80 mV .The pipette solution contained 140mM KCl, 5 mM K₂HPO₄, 5 mM KH₂PO₄, 0.3 mM CaCl₂, 1 mM MgCl₂, pH 7.2 withKOH. The bath solution contained 130 mM KCl, 5 mM K₂EDTA, 5 mM K₂HPO₄, 5mM KH₂PO₄, pH 7.2 with KOH.

For the single channel I-V curve shown in Figure S1D inset, each datapoint represents the current difference at a given voltage associatedwith the opening of a single channel.

REFERENCES

-   1. J. P. Wong, E. Reboul, R. S. Molday, J. Kast, J Proteome Res 8,    2388 (May, 2009).-   2. Z. Otwinowski, W. Minor, Methods Enzymol. 276, 307 (1997).-   3. N. Collaborative Computational Project, Acta Cryst. D50, 60    (1994).-   4. A. Vagin, A. Teplyakov, Acta Crystallogr. D. Biol. Crystallogr.    56 Pt 12:1622-4., 1622 (2000).-   5. T. A. Jones, J. Y. Zola, S. W. Cowan, M. Kjeldgaard, Acta Cryst.    A47, 110 (1991).-   6. A. T. Brunger et al., Acta Cryst. D54, 905 (1998).-   7. W. L. DeLano, DeLano Scientific, Palo Alto, Calif., USA.    http://www.pymol.org, (2002).

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

It is further to be understood that all values are approximate, and areprovided for description.

All patents, applications, publications, test methods, literature, andother materials cited herein are hereby incorporated by reference.

TABLE S1 Crystallographic data and refinement statistics Data CollectionData set Native Rb⁺ (650 mM) Sr²⁺ (10 mM) Sr²⁺ (200 mM) Eu³⁺ (10 mM)Space group I4 I4 I4 I4 I4 Lattice constants (Å) a = b = 84.018, a = b =82.714, a = b = 82.806, a = b = 83.268, a = b = 84.221, c = 196.121 c=196.142 c = 195.605 c = 197.143 c = 195.915 α = β = γ = 90° α = β = γ =90° α = β = γ = 90° α = β = γ = 90° α = β = γ = 90° Source APS 24ID-CBNL X29 BNL X29 BNL X29 BNL X29 Wavelength (Å) 0.97949 1.0809 1.08091.0809 1.5222 Resolution (Å) 50-3.1 50-4.0 50-3.3 50-3.8 50-6.0Total/unique observations 65,452/12,153 25,333/5,231 60,844/9,91031,462/6,571 5,649/1,517 I/sigma (I) ^(a) 33.1 (23) 20.0 (1.8) 24.3(2.4) 20.8 (2.7) 24.7 (1.6) Redundancy ^(a) 5.4 (4.6) 4.8 (4.3) 6.1(5.9) 4.8 (4.7) 3.7 (1.4) Completeness (%) ^(a) 99.7 (99.9) 93.3 (83.8)99.8 (100.0) 99.7 (100.0) 87.9 (49.4) R_(sym) (%) ^(a,b) 6.8 (73.6) 10.1(78.3) 11.5 (89.4) 9.6 (69.3) 7.0 (35.7) Model refinement Resolution (Å)50-3.1 50-4.0 50-3.3 50-3.8 50-6.0 Number of reflections 12,122 (609)5,221 (272) 9,895 (504) 6,569 (346) 1,517 (76) R_(work)/R_(free)24.4/27.2 29.6/35.0 24.9/28.6 25.8/30.4 36.5/39.9 R.m.s. deviation ofbond length (Å) 0.010 R.m.s. deviation of bond angles (°) 1.59 Proteinatoms/K⁺ ions 2.530/5 Mean B value 112.1 Ramachandran plot ^(c)76.0/21.9/2.1 R.m.s., root mean-squared. ^(a) Number in the parenthesesrepresents statistics for data in the highest resolution shell. ^(b)R_(sym) = Σ|I_(i) − <I_(i)>|/ΣI_(i), where <I_(i)> is the averageintensity of symmetry equivalent reflections. ^(c) The three numbersrepresent the percentage of residues in most favored/additionallyallowed/generously allowed regions.

SEQUENCES: 1. human Kir 1.1- variable portion of turret regionPEFHPSANHTP 2. human Kir 1.2- variable portion of turret regionLELDPPANHTP 3. human Kir 2.1- variable portion of turret region DASKEGKA4. human Kir 2.2- variable portion of turret region EPAEGRGRTP5. human Kir 2.3- variable portion of turret regionEASPGVPAAGGPAAGGGGAAPVAPKP6. human Kir 2.4- variable portion of turret region AAPPPPAP7. human Kir 3.1- variable portion of turret region NKAHVGNYTP8. human Kir 3.4- variable portion of turret region DHVGDQEWIP9. human Kir 6.1- variable portion of turret region YAYMEKSGMEKSGLESTV10. human Kir 6.2- variable portion of turret region APSEGTAEP11. human Kir 7.1- variable portion of turret region ELDHDAPPENHTI12. chicken Kir 2.1- variable portion of turret region ENQENNKP13. chicken Kir 2.2- variable portion of turret region ENPGGDDTFKP14. human Kir 1.1- amino acid sequence    1MNASSRNVFD TLIRVLTESM FKHLRKWVVT REFGHSRQRA RLVSKDGRCN IEFGNVEAQS   61RFIFFVDIWT TVLDLKWRYK MTIFITAFLG SWEFFGLLWY AVAYIHKDLP EFHPSANHTP  121CVENINGLTS AFLFSLETQV TIGYGFRCVT EQCATAIFLL IFQSILGVII NSFMCGAILA  181KISRPKKRAK TITFSKNAVI SKRGGKLCLL IRVANLRKSL LIGSHIYGKL LKTTVTPEGE  241TIILDQININ FVVDAGNENL FFISPLTIYH VIDHNSPFFH MAAETLLQQD FELVVFLDGT  301VESTSATCQV RTSYVPEEVL WGYRFAPLVS KTKEGKYRVD FHNFSKTVEV ETPHCAMCLY  361NEKDVRARMK RGYDNPNFIL SEVNETDDTK M15. human Kir 1.2- amino acid sequence    1MTSVAKVYYS QTTQTESRPL MGPGIRRRRV LTKDGRSNVR MEHIADKRFL YLKDLWTTFI   61DMQWRYKLLL FSATFAGTWF LFGVVWYLVA VAHGDLLELD PPANHTPCVV QVHTLTGAFL  121FSLESQTTIG YGFRYISEEC PLAIVLLIAQ LVLTTILEIF ITGTFLAKIA RPKKRAETIR  181FSQHAVVASH NGKFCLMIRV ANMRKSLLIG CQVTGKLLQT HQTKEGENIR LNQVNVTFQV  241DTASDSPFLI LPLTFYHVVD ETSPLKDLPL RSGEGDFELV LILSGTVEST SATCQVRTSY  301LPEEILWGYE FTPAISLSAS GKYIADFSLF DQVVKVASPS GLRDSTVRYG DPEKLKLEES  361LREQAEKEGS ALSVRISNV 16. human Kir 2.1- amino acid sequence    1MGSVRTNRYS IVSSEEDGMK LATMAVANGF GNGKSKVHTR QQCRSRFVKK DGHCNVQFIN   61VGEKGQRYLA DTFTTCVDIR WRWMLVIFCL AFVLSWLFFG CVFWLIALLH GDLDASKFGK  121ACVSEVNSFT AAFLFSIETQ TTIGYGFRCV TDECPIAVFM VVFQSIVGCI IDAFIIGAVM  181AKMAKPKKRN ETLVFSHNAV IAMRDGKLCL MWRVGNLRKS HLVEAHVRAQ LLKSRITSEG  241EYIPLDQIDI NVGFDSGIDR IFLVSPITIV HEIDED5PLY DLSKQDIDNA DFEIVVILEG  301MVEATAMITQ CRSSYLANEI LVGHRYEPVL FEEKHYYKVD YSRFHKTYEV PNTPLCSARD  361LAEKKYILSN ANSFCYENEV ALTSKEEDDS ENGVPESTST DTPPDIDLHN QASVPLEPRP  421LRRESEI 17. human Kir 2.2- amino acid sequence    1MTAASRANPY SIVSSEEDGL HLVTMSGANG FGNGKVHTRR RCRNRFVKKN GQCNIEFANM   61DEKSQRYLAD MFTTCVDIRW RYMLLIFSLA FLASWLLEGI IFWVIAVAHG DLEPAEGRGR  121TPCVMQVHGE MAAFLPSIET QTTIGYGLRC VTEECPVAVF MVVAQSIVGC IIDSFMIGAI  181MAKMARPKKR AQTLLFSHNA VVALRDGKLC LMWRVGNLRK SHIVEAHVRA QLIKPRVTEE  241GEYIPLDQID IDVGFDKGLD RIFLVSPITI LHEIDEASPL FGISRQDLET DDFEIVVILE  301GMVEATAMTT QARSSYLANE ILWGHRFEPV LFEEKNQYKI DYSHFHKTYE VPSTPRCSAX  361DLVENKFLLP SANSFCYENE LAFLSRDEED EADGDQDGRS RDGLSPQARH DFDRLQAGGG  421VLEQRPYRRE SEI 18. human Kir 2.3- amino acid sequence    1MHGHSRNGQA HVPRRKRRNR FVKKNGQCNV YFANLSNKSQ RYMADIFTTC VDTRWRYMLM   61IFSAAFLVSW LFFGLLFWCI AFFHGDLEAS PGVPAAGGPA AGGGGAAPVA PKPCIMHVNG  121FLCAFLFSVE TQTTIGYGFR CVTEECPLAV IAVVVQSIVG CVIDSFMIGT IMAKMARPKK  181RAQTLLFSHH AVISVRDGKL CLMWRVGNLR KSHIVEAHVR AQLIKPYMTQ EGEYLPLDQR  241DLNVGYDIGL DRIFLVSPII IVHEIDEDSP LYGMGKEELE SEDFEIVVIL EGMVEATAMT  301TQARSSYLAS EILWGHRFEP VVFEEKSHYK VDYSRFHKTY EVAGTPCCSA RELQESKITV  361LPAPPPPPSA FCYENELALM SQEEEEMEEE AAAAAAVAAG LGLEAGSKEE AGIIRMLEFG  421SHLDLERMQA SLPLDNISYR RESAI 19. human Kir 2.4- sequence    1MGLARALRRL SGALDSGDSR AGDEEEAGPG LCRNGWAPAP VQSPVGRRRG RFVKKDGHCN   61VREVNLGGQG ARYLSDLFTT CVDVRWRWMC LLFSCSFLAS WLLFGLAFWL IASLHGDLAA  121PPPPAPCFSH VASFLAAFLF ALETQTSIGY GVRSVTEECP AAVAAVVLQC IAGCVLDAFV  181VGAVMAXMAK PKKRNEILVF SENAVVALRD HRLCLMWRVG NLRRSHLVEA HVRAQLLQPR  241VTPEGEYIPL DHQDVDVGFD GGTDRIFLVS PITIVHEIDS ASPLYELGRA ELARADFELV  301VILEGMVEAT AMTTQCRSSY LPGELLWGHR FEPVLFQRGS QYEVDYRHFH RTYEVPGTPV  361CSAKELDERA EQASHSLKSS FPGSLTAFCY ENELALSCCQ EEDEDDETEE GNGVETEDGA  421ASPRVLTPTL ALTLPP 20. human Kir 3.1- amino acid sequence    1MSALRRKFGD DYQVVTTSSS GSGLQPQGPG QDPQQQLVPK KKRQRFVDKN GRCNVQHGNL   61GSETSRYLSD LFTTLVDLKW RWNLFIFILT YTVAWLFMAS MNWVIAYTRG DLNKAHVGNY  121TPCVANVYNF PSAFLEFIET EATIGYGYRY ITDKCPEGII LFLFQSILGS IVDAFLIGCM  181FIKMSQPKKR AETLMFSEHA VISMRDGKLT LMERVGNERN SHMVSAQIRC KLLKSRQTPE  241GEFLPLDQLE LDVGFSTGAD QLFLVSPLTI CHVIDAKSPF YDLSQRSMQT EQFEIVVILE  301GIVETTGMTC QARTSYTEDE VLWGHRFFPV ISLEEGFFKV DYSQFHATFE VPTPPYSVKE  361QEEMLLMSSP LIAPAITNSK ERHNSVECLD GLDDITITLP SKLQKITGRE DFPKKLLRMS  421STTSEKAYSL GDLPMKLQRI SSVPGNSEEK LVSKTTKMLS DPMSQSVADL PPKLQKMAGG  481AARMEGNLPA KLRKMNSDRF T 21. human Kir 3.4- amino acid sequence    1MAGDSRNAMN QDKEIGVTPW DPKKIPKQAR DYVPIATDRT RLLAEGKKPR QRYNEKSGKC   61NVHHGNVQET YRYLSDLFTT LVDLKWRFNL LVFTMVYTVT WLFEGFIWWL IAYIRGDLDH  121VGDQEWIPCV ENLSGFVSAF LFSIETETTI GYGFRVITEK CPEGIILLLV QAILGSIVNA  181FMVGCMFVKI SQPKKRAETL MESNNAVISM RDEKLCLMFR VGDLRNSHIV EASIRAKLIK  241SRQTKEGEFI PLNQTDINVG FDTGDDRLFL VSPLIISHEI NQKSPFWEMS QAQLHQEEFE  301VVVILEGMVE ATGMTCQARS SYMDTEVLWG HRFTPVLTLE KGFYEVDYNT FHDTYETNTP  361SCCAKELAEM KREGRLLQYL PSPPLLGGCA EAGLDAEAEQ NEEDEPKGLG GSREARGSV22. human Kir 4.1- amino acid sequence    1MTSVARVYYS QTTQTESRPL MGPGIRRRRV LTKDGRSNVR MEHIADKRFL YLKDLWTTFI   61DMQWRYKLLL FSATFAGTWF LFGVVWYLVA VAHGDLLELD PPANHTPCVV QVHTLTGAFL  121FSLESQTTIG YGFRYISEEC PLAIVLLIAQ LVLTTILEIF ITGTFLAKIA RPKKRAETIR  181FSQHAVVASH NGKPCLMIRV ANMRKSLLIG CQVTGKLLQT HQTKEGENIR LNQVNVTFQV  241DTASDSPFLI LPLTFYHVVD ETSPLKDLPL RSGEGDFELV LILSGTVEST SATCQVRTSY  301LPEEILWGYE FTPAISLSAS GKYIADFSLF DQVVKVASPS GLRDSTVRYG DPEKLKLEES  361LREQAEKEGS ALSVRISNV 23. human Kir 4.2- amino acid sequence    1MDAIHIGMSS TPLVKHTAGA GLKANRPRVM SKSGHSNVRI DKVDGIYLLY LQDLWTTVID   61NKWRYKLTLF AATFVMTWFL FGVIYYAIAF IHGDLEPGEP ISNHTPCINE VDSLTGAFLF  121SLESQTTIGY GVRSITEECP HAIFILVAQL VITTLIEIFI TGTFLAKIAR PKKRAETIKF  181SHCAVITXQN GKLCLVIQVA NMRKSLLIQC QLSGKLLQTH VTKEGERILL NQATVKFHVD  241SSSESPFLIL PMTFYHVLDE TSPLRDLTPQ NLKEKEFELV VLLNATVEST SAVCQSRTSY  301IPEEIYWGFE FVPVVSLSKN GKYVADFSQF EQIRKSPDCT FYCADSEKQQ LEEKYRQEDQ  361RERELRTLLL QQSNV 24. human Kir 5.1- amino acid sequence    1MSYYGSSYHI INADAKYPGY PPEHIIAEKR RARRRLLHKD GSCNVYFKHI FGEWGSYVVD   61IFTTLVDTKW RHMEVIFSLS YILSWLIFGS VFWLIAFHHG OLLNDPDITP CVDNVHSFTG  121AFLFSLETQT TIGYGYRCVT EECSVAVLMV ILQSILSCII NTFIIGAALA KMATARKRAQ  181TIRESYFALI GMRDGKLCLM WRIGDFRPNH VVEGTVRAQL LRYTEDSEGR MTMAFKDLKL  241VNDQIILVTP VTIVHEIDHE SPLYALDRKA VAKDNFEILV TFIYTGDSTG TSHQSRSSYV  301PREILWOHRF NOVLEVKRKY YKVNCLQFEG SVEVYAPFCS AKQLDWKDQQ LHIEKAPPVR  361ESCTSDTKAR RRSFSAVAIV SSCENPEETT TSATHEYRET PYQKALLTLN RISVESQM25. human Kir 6.1- amino acid sequence    1MLARKSIIPE EYVLARIAAE NLRKPRIRDR LPKARFIAKS GACNLAHKNI REQGRFLQDI   61FTTLVDLKWR HTLVIFTMSF LCSWLLFAIM WWLVAFAHGD IYAYMEKSGM EKSGLFSTVC  121VTNVRSFTSA FLFSIEVQVT IGFGGRMMTE ECPLAITVLI LQNIVGLIIN AVMLGCIFMK  181TAQAHRRAET LIFSRHAVIA VRNGKLCFMF RVGDLRKSMI ISASVRIQVV KKTTTPEGEV  241VPIHQLDIPV DNPIESNNIF LVAPLIICHV IDKRSPLYDI SATDLANQDL EVIVILEGVV  301ETTGITTQAR TSYIAEEIQW GHRFVSIVTE EEGVYSVDYS KFGNTVKVAA PRCSARELDE  361KPSILIQTLQ KSELSHQNSL RKRNSMRRNN SMRRNNSIRR NNSSLMVPKV QFMTPEGNQN  421TSES 26. human Kir 6.2- amino acid sequence    1MLSRKGIIPE EYVLTRLAED PAKPRYRARQ RRARFVSKKG NCNVAHKNIR EQGRFLQDVF   61TTLVDLKWPH TLLIFTMSFL CSWLLFAMAW WLEAFAHGDL APSEGTAEPC VTSIHSFSSA  121FLFSIEVQVT IGFGGRMVTE ECPLAILILI VQNIVGLMIN AIMLGCIFMK TAQAHRRAET  181LIFSKHAVIA LRHGRLCFML RVGDLRKSMI ISATIHMQVV RKTTSPEGEV VPLHQVDIPM  241ENGVGGNSIF LVAPLIIYHV IDANSPLYDL APSDLHHHQD LEIIVILEGV VETTGITTQA  301RTSYLADEIL WGQRFVPIVA EEDGRYSVDY SKFGNTVKVP TPLCTARQLD EDHSLLEALT  361LASARGPLRK RSVPMAKAKP KFSISPDSLS 27. human Kir 7.1- amino acid sequence   1 mdssnckvia pllsgryrrm vtkdghstlq mdgagrglay lrdawgilmd mrwrwmmlvf  61 sasfvvhwlv favlwyvlae mngdleldhd appenhticv kyitsftaaf sfsletqlti 121 gygtmfpsgd cpsaiallai gmllglmlea fitgafvaki arpknrafsi rftdtavvah 181 mdgkpnlifq vantrpsplt svrvsavlyq erengklygt svdfhldgis sdecpffifp 241 ltyyhsitps sp1atllghe npshfelvvf lsamgegtge icgrrtsylp seimlhhcfa 301 slltrgskge ygikmenfdk typefptply skspnrtdld ihinggsidn fqisetglte28. chicken Kir 2.1- amino acid sequence    1MGSVRTNRYS IVSSEEDGMK LATMAVANGF GNGKSKVHTR QQCRSRFVXK DGHCNVQFIN   61VGEKGQRYLA DIFTTCVDIR WRWMLVIFCL TEILSWLFFG CVFWLIALLH GDLENQENNK  121PCVSQVSSFT AAFLFSIETQ TTIGYGFRCV TDECPIAVFM VVFQSIVGCI IDAFIIGAVM  181AKMAKPKKRN ETLVFSHNAV VAMRDGKLCL MWRVGNLRKS HLVEAHVRAQ LLKSRITSEG  241EYIPLDEIDI NVGFDSGIDR IFLVSPITIV HEIDEDSPLY DLSKQDMDNA DFEIVVILEG  301MVEATAMTTQ CRSSYLANEI LWGHRYEPVL FEEKNYYKVD YSRFHKTYEV PNTPICSARD  361LAEKKYILSN ANSFCYENEV ALTSKEEDEI DTGVPESTST DTHPDMDHHN QAGVPLEPRP  421LRRESEI 29. chicken Kir 2.2- amino acid sequence    1mtagrvnpys ivsseedglr lttmpgingf gngkihtrrk crnrfvkkng qcnveftnmd   61dkpqryiadm fttcvdirwr ymlllfslaf lvswllfgli fwlialihgd lenpggddtf  121kpcvlqvngf vaallfsiet qttigygfrc vteecplavf mvvvqsivgc iidsfmigai  181makmarpkkr aqtllfshna vvamrdgklc lmwrvgnlrk shiveahvra qlikpritee  241geyipldgid idvgfdkgld riflvspiti lheinedspl fgisrqdlet ddfeivvile  301gmveatamtt qarssylase ilwghrfepv lfeeknqykv dyshfhktye vpstprcsak  361dlvenkfllp stnsfcyene lafmsrdede edddsrgldd lspdnrhefd rlqatialdq  421rsyrresei 30. human Kir 1.1- cDNA    1atgaatgctt ccagtcggaa tgtgtttgac acgttgatca gggtgttgac agaaagtatg   61ttcaaacatc ttcggaaatg ggtcgtcact cgcttttttg ggcattctcg gcaaagagca  121aggctagtct ccaaagatgg aaggtgcaac atagaatttg gcaatgtgga ggcacagtca  181aggtttatat tctttgtgga catctggaca acggtacttg acctcaagtg gagatacaaa  241atgaccattt tcatcacagc cttcttgggg agttggtttt tctttggtct cctgtggtat  301gcagtagcgt acattcacaa agacctcccg gaattccatc cttctgccaa tcacactccc  361tgtgtggaga atattaatgg cttgacctca gcttttctgt tttctctgga gactcaagtg  421accattggat atggattcag gtgtgtgaca gaacagtgtg ccactgccat ttttctgctt  481atctttcagt ctatacttgg agttataatc aattctttca tgtgtggggc catcttagcc  541aagatctcca ggcccaaaaa acgtgccaag accattacgt tcagcaagaa cgcagtgatc  601agcaaacggg gagggaagct ttgcctccta atccgagcgg ctaatctcag gaagagcctt  661cttattggca gtcacattta tggaaagctt ctgaagacca cagtcactcc tgaaggagag  721accattattt tggaccagat caatatcaac tttgtagttg acgctgggaa tgaaaattta  781ttcttcatct ccccattgac aatttaccat gtcattgatc acaacagccc tttcttccac  841atggcagcgg agacccttct ccagcaggac tttgaattag tggtgttttt agatggcaca  901gtggagtcca ccagtgctac ctgccaagtc cggacatcct atgtcccaga ggaggtgctt  961tggggctacc gttttgctcc catagtatcc aagacaaagg aagggaaata ccgagtggat 1021ttccataact ttagcaagac agtggaagtg gagacccctc actgtgccat gtgcctttat 1081aatgagaaag atgttagagc caggatgaag agaggctatg acaaccccaa cttcatcttg 1141tcagaagtca atgaaacaga tgacaccaaa atgtaa 31. human Kir 1.2- cDNA    1cttttctgat cccagctccg ggtttaagag tcctggcacg gcccgtcgca cagctctgct   61cctaactcct gcccgccccg tccgtccatc tgtcccgctg ccccgcggcc catccaaggg  121gccactccac ctcggaccca agatgacgtc agttgccaag gtgtattaca gtcagaccac  181tcagacagaa agccggcccc taatgggccc agggatacga cggcggagag tcctgacaaa  241agatggtcgc agcaacgtga gaatggagca cattgccgac aagcgcttcc tctacctcaa  301ggacctgtgg acaaccttca ttgacatgca gtggcgctac aagcttctgc tcttctctgc  361gacctttgca ggcacatggt tcctctttgt cgtggtgtgg tatctggtag ctgtggcaca  421tggggacctg ctggagctgg accccccggc caaccacacc ccctgtgtgg tacaggtgca  481cacactcact ggagccttcc tcttctccct tgaatcccaa accaccattg gctatggctt  541ccgctacatc agtgaggaat gtccactagc cattgtgctt cttattgccc agctggtgct  601caccaccatc ctggaaatct tcatcacagg taccttcctg gcgaagattg cccggcccaa  661gaagcgggct gagaccattc gtttcagcca gcatgcagtt gtggcctccc acaatggcaa  721gccctgcctc atgatccgag ttgccaatat gcgcaaaagc ctcctcattg gctgccaggt  781gacaggaaaa ctgcttcaga cccaccaaac caaggaaggg gagaacaccc ggctcaacca  841ggtcaatgtg actttccaag tagacacagc ccctgacagc cccttcctta ttctacccct  901taccttctat catgtggtag atgagaccag tcccttgaaa gatctccctc ttcgcagcgg  961tgagggtgac tttgagctgg tgctgatcct aagtgggaca gtggagtcca ccagtgccac 1021ctgtcaggtg cgcacttcct acctgccaga ggagatcctt tggggctacg agttcacacc 1081tgccatctca ctgtcagcca gtggtaaata catagctgac tttagccttt ttgaccaagt 1141tgtgaaagtg gcctctccta gtggcctccg tgacagcact gtacgctacg gagaccctga 1201aaagctcaag ttggaggagt cattaaggga gcaagctgag aaggagggca gtgcccttag 1261tgtgcgcatc agcaatgtct ga 32. human Kir 2.1- cDNA    1atgggcagtg tgcgaaccaa ccgctacagc atcgtctctt cagaagaaga cggtatgaag   61ttggccacca tggcagttgc aaatggcttt gggaacggga agagtaaagt ccacacccga  121caacagtgca ggagccgctt tgtgaagaaa gatggccact gtaatgttca gttcatcaat  181gtgggtgaga aggggcaacg gtacctcgca gacatcttca ccacgtgtgt ggacattcgc  241tggcggtgga tgctggttat cttctgcctg gctttcgtcc tgtcatggct gttttttggc  301Lgtgtgcttt ggttgatagc tctgctccat ggggacctgg atgcatccaa agagggcaaa  361gcttgtgtgt ccgaggtcaa cagcttcacg gctgccttcc tcttctccat tgagacccag  421acaaccatag gctatggttt cagatgtgtc acggatgaat gcccaattgc tgttttcatg  481gtggtgttcc agtcaatcgt gggctgcatc atcgatgctt tcatcattgg cgcagtcatg  541gccaagatgg caaagccaaa gaagagaaac gagactcttg tcttcagtca caatgccgtg  601attgccatga gagacggcaa gctgtgtttg atgtggcgag tgggcaatct tcggaaaagc  661cacttggtgg aagctcatgt tcgagcacag ctcctcaaat ccagaattac ttctgaaggg  721gagtatatcc ctctggatca aatagacatc aatgttgggt ttgagagcgg aatcgatcgt  781atatttctgg tgtccccaat cactatagtc catgaaatag atgaagacag tcctttatat  841gatttgagta aacaggacat tgacaacgca gactttgaaa tcgtggtcat actggaaggc  901atggtggaag ccactgccat gacgacacag tgccgtagct cttatctagc aaatgaaatc  961ctgtggggcc accgctatga gcctgtgctc tttgaagaga agcactacta caaagtggac 1021tattccaggt tccacaaaac ttacgaagtc cccaacactc ccctttgtag tgccagagac 1081ttagcagaaa agaaatatat cctctcaaat gcaaattcat tttgctatga aaatgaagtt 1141gccctcacaa gcaaagagga agacgacagt gaaaatggag ttccagaaag cactagtacg 1201gacacgcccc ctgacataga ccttcacaac caggcaagtg tacctctaga gcccaggccc 1261ttacggcgag agtcggagat atga 33. human Kir 2.2- cDNA    1atgaccgcgg ccagccgggc caacccctac agcatcgtgt catcggagga ggacgggctg   61cacctggtca ccatgtcggg cgccaacggc ttcggcaacg gcaaggtgca cacgcgccgc  121aggtgccgca accgcttcgt caagaagaat ggccagtgca acattgagtt cgccaacatg  181gacgagaagt cacagcgcta cctggctgac atgtccacca cctgtgcgga catccgctgg  241cggtacatgc tgctcatctt ctcgctggcc ttccttgcct cctggctgct gttcggcatc  301atcttctggg tcatcgcggt ggcacacggt gacctggagc cggctgaggg ccggggccgc  361acaccctgtg tgatgcaggt gcacggcttc atggcggcct tcctcttctc catcgagacg  421cagaccacca tcggctacgg gctgcgctgt gtgacggagg agtgcccggt ggccgtcttc  481atggtggtgg cccagtccat cgtgggctgc atcatcgact cctccatgat tggtgccatc  541atggccaaga tggcaaggcc caagaagcgg gcacagacgc tgctgttgag ccacaacgcc  601gtggtggccc tgcgtgacgg caagctctgc ctcatgtggc gtgtgggtaa cctgcgcaag  661agccacattg tggaggccca tgtgcgcgcg cagctcatca agccgcgggt caccgaggag  721ggcgagtaca tcccgctgga ccagatcgac atcgatgtgg gcttcgacaa gggcctggac  781cgcatctttc tggtgtcgcc catcaccatc ttgcatgaga ttgacgaggc caggccgctc  841ttcggcatca gccggcagga cctggagacg gacgactttg agatcgtggt catcctggaa  901ggcatggtgg aggccacagc catgaccacc caggcccgca gctcctacct ggccaatgag  961atcttctggg gtcaccgctt tgagcccgtg ctcttcgagg agaagaacca gtacaagatt 1021gactactcgc acttccacaa gacctatgag gtgccctcta cgccccgctg cagtgcgaag 1081gatctggtag agaacaagtt cctgctgccc agcgccaact ccttctgcta cgagaacgag 1141ctggccttcc tgagccgtga cgaggaggat gaggcggacg gagaccagga cggccgaagc 1201cgggacggcc tcagccccca ggccaggcat gactttgaca gactccaggc tggcggcggg 1261gacctggagc agcggcccta cagacgggag tcagagatct ga 34. human Kir 2.3- cDNA   1 atgcacggac acagccgcaa cggccaggcc cacgtgcccc ggcggaagcg ccgcaaccgc  61 ttcgtcaaga agaacggcca atgcaacgtg tacttcgcca acctgagcaa caagtcgcag 121 cgctacatgg cggacatctt caccacctgc gtggacacgc gctggcgcta catgctcatg 181 atcttctccg cggccttcct tgtctcctgg ctctttttcg gcctcctctt ctggtgtatc 241 gccttcttcc acggtgacct ggaggccagc ccaggggtgc ctgcggcggg gggcccggcg 301 gcgggtggtg gcggaggagc cccggtggcc cccaagccct gcatcatgca cgtgaacggc 361 ttcctgggtg ccttcctgtt ctcggtggag acgcagacga ccatcggcta tgggttccgg 421 tgcgtgacag aggagtgccc gctggcagtc atcgctgtgg tggtccagtc catcgtgggc 481 tgcgtcatcg actccttcat gattggcacc atcatggcca agatggcgcg gcccaagaag 541 cgggcgcaga cgttgctgtt cagccaccac gcggtcattt cggtgcgcga cggcaagctc 601 tgcctcatgt ggcgcgtggg caacctgcgc aagagccaca ttgtggaggc ccacgtgcgg 661 gcccagctca tcaagccata catgacccag gagggcgagt acctgcccct ggaccagcgg 721 gacctcaacg tgggctatga catcggcctg gaccgcatct tcctggtgtc gcccatcatc 781 attgtccacg agatcgacga ggacagcccg ctttatggca tgggcaagga ggagctggag 841 tcggaggact ttgagatcgt ggtcatcctg gagggcatgg tggaggccac ggccatgacc 901 acccaggccc gcagctccta cctggccagc gagatcctgt ggggccaccg ctttgagcct 961 gtggtcttcg aggagaagag ccactacaag gtggactact cacgttttca caagacctac1021 gaggtggccg gcacgccctg ctgctcggcc cgggagctgc aggagagtaa gatcaccgtg1081 ctgcccgccc caccgccccc tcccagtgcc ttctgctacg agaacgagct ggcccttatg1141 agccaggagg aagaggagat ggaggaggag gcagctgcgg cggccgcggt ggccgcaggc1201 ctgggcctgg aggcgggttc caaggaggag gcgggcatca tccggatgct ggagttcggc1261 agccacctgg acctggagcg catgcaggct tccctcccgc tggacaacat ctcctaccgc1321 agggagtctg ccatctga 35. human Kir 2.4- cDNA    1atgggcctgg ccagggccct acgccgcctc agcggcgccc tggattcggg agacagccgg   61gcgggcgatg aagaggaggc cgggcccggg ttgtgccgca acgggtgggc gccggcaccg  121gtgcagtcac ccgtgggccg gcgccgcggt cgcttcgtca agaaagacgg gcactgcaac  181gtgcgtttcg taaacctggg tggccagggc gcgcgctacc tgagcgacct gttcaccaca  241tgcgtggacg tgcgctggcg ctggatgtgc ctgctcttct cctgctcctt cctcgcctcc  301tggctgctct tcggcctggc cttctggctc attgcctcgc tgcacggcga cctggccgcc  361ccgccaccgc ccgcgccctg cttctcacac gtggccagct tcctggccgc cttcctcttc  421gcgctggaga cgcagacgtc catcggctac ggcgtgcgca gcgtcaccga ggagtgcccg  481gccgctgtgg ccgccgtggt gctgcagtgc attgccggct gcgtgctcga cgccttcgtc  541gtgggtgctg tcatggccaa gatggccaaa cccaagaagc gcaacgagac gctggtcttc  601agcgagaacg ccgtcgtggc gctgcgcgac caccgcctct gcctcatgtg gcgcgtcggc  661aacctgcgcc gcagccacct ggtcgagacc cacgtgcgtg cccagctgct gcagccccgt  721gtgaccccag agggtgagta catcccgctg gaccaccagg atgtggatgt gggctttgat  781ggaggcaccg atcgtatctt cctcgtgtcc cccatcacca tcgtccatga gatcgactct  841gccagtcctc tgtatgagct aggacgtgcc gagctggcca gggctgactt tgagctggtg  901gtcattctcg aggggatggt tgaggccaca gccatgacca cacagtgtcg ctcgtcctac  961ctccctggtg aactgctctg gggccatcgt tttgagccag ttctcttcca gcgtggctcc 1021cagtatgagg tcgactatcg ccacttccat cgcacttatg aggtcccagg gacaccggtc 1081tgcagtgcta aggagctgga tgaacgggca gagcaggctt cccacagcct caagtctagt 1141ttccccggct ctctgactgc attttgttat gagaatgaac ttgctctgag ctgctgccag 1201gaggaagatg aggacgatga gactgaggaa gggaatgggg tggaaacaga agatggggct 1261gctagccccc gagttctcac accaaccctg gcgctgaccc tgcctccatg a36. human Kir 3.1- cDNA    1atgtctgcac tccgaaggaa atttggggac gattatcagg tagtgaccac atcgtccagc   61ggctcgggct tgcagcccca ggggccaggc caggaccctc aggaggagct tgtgcccaag  121aagaagcggc agcggttcgt ggacaagaac ggccggtgca atgtacagca cggcaacctg  181ggcagcgaga caagccgcta cctctcggac ctcttcacca cgctggtgga cctcaagtgg  241cgctggaacc tcttcatctt cattctcacc tacaccgtgg cctggctttt catggcgtcc  301atgtggtggg tgatcgccta cactcggggc gacctgaaca aagcccacgt cggtaactac  361acgccttgcg tggccaatgt ctataacttc ccttctgcct tcctcttctt catcgagacg  421gaggccacca tcggctatgg ctaccgatac atcacagaca agtgccccga gggcatcatc  481ctcttcctct tccagtccat cctgggctcc atcgtggacg ccttcctcat cggctgcatg  541ttcatcaaga tgtcccagcc caagaagcgc gccgagaccc tcatgttcag cgagcacgcg  601gtgatctcca tgagggacgg aaaactcacg cttatgttcc gggtgggcaa cctgcgcaac  661agccacatgg tctccgcgca gattcgctgc aagctgctca aatctcggca gacacctgag  721ggtgagttcc ttccccttga ccaacttgaa ctggatgtag gttttagtac aggggcagat  781caactttttc ttgtgtcccc cctcacaatt tgccacgtga tcgatgccaa aagccccttt  841tatgacctat cccagcgaag catgcaaact gaacagttcg agattgtcgt catcctagaa  901ggcattgtgg aaacaactgg gatgacttgt caagctcgaa catcatatac tgaagatgaa  961gttctttggg gtcatcgttt ttttcctgta atttccttag aagagggatt ctttaaagtt 1021gattactccc agttccatgc aacatttgaa gtccccaccc caccttacag tgtgaaagag 1081caggaggaaa tgcttctcat gtcgtcccct ttaatagcac cagccataac taacagcaaa 1141gaaagacata attctgtgga atgcttagat ggactagatg atattactac aaaactacca 1201tctaagctgc agaaaattac tggaagagaa gactttccca aaaaactctt gaggatgagt 1261tctacaactt cagaaaaagc ctacagcttg ggagacttgc ccatgaaact tcaacgaata 1321agttcagttc cgggcaactc agaagaaaaa ctggtatcta aaaccaccaa gatgttatct 1381gatcccatga gccagtctgt ggctgatttg ccaccaaagc ttcaaaagat ggctggagga 1441gcagctagga tggaagggaa ccttccagcc aaattaagaa aaatgaactc tgatcgcttc 1501acataa 37. human Kir 3.4- cDNA    1atggctggcg attctaggaa tgccatgaac caggacatgg agattggagt cactccctgg   61gaccccaaga agattccaaa acaggcccgc gattatgtcc ccattgccac agaccgtacg  121cgcctgctgg ccgagggcaa gaagccacgc cagcgctaca tggagaagag tggcaagtgc  181aacgtgcacc acggcaacgt ccaggagacc taccggtacc tgagtgacct cttcaccacc  241ctggtggacc tcaagtggcg cttcaacttg ctcgtcttca ccatggttta cactgtcacc  301tggctgttct tcggcttcat ttggtggctc attgcttata tccggggtga cctggaccat  361gttggcgacc aagagtggat tccttgtgtt gaaaacctca gtggcttcgt gtccgctttc  421ctgttctcca ttgagaccga aacaaccatt gggtatggct tccgagtcat cacagagaag  481tgtccagagg ggattatact cctcttggtc caggccaLcc tgggctccat cgtcaatgcc  541ttcatggtgg ggtgcatgtt tgtcaagatc agccagccca agaagagagc ggagaccctc  601atgttttcca acaacgcagt catctccatg cgggacgaga agctgtgcct catgttccgg  661gttggcgacc tccgcaactC CCacatcgtg gaggcctcca tccgggccaa gctcatcaag  721tcccggcaga ccaaagaggg ggagttcatc cccctgaacc agacagacat caacgtgggc  781tttgacacgg gcgacgaccg cctcttcctt gtgtctcctc tgatcatctc ccatgagatc  841aaccagaaga gccctttctg ggagatgtct caggctcagc tgcatcagga agagtttgaa  901gttgtggtca ttctagaagg gatggtggaa gccacaggca tgacctgcca agcccggagc  961tcctacatgg atacagaggt gctctggggc caccgattca ccaccatcct caccttggaa 1021aagggcttct atgaggtgga ctacaacacc ttccatgata cctatgagac caacacaccc 1081agctgctgtg ccaaggagct ggcagaaatg aagagggaag gccggctcct ccagtacctc 1141cccagccccc cactgctggg gggctgtgct gaggcagggc tggatgcaga ggctgagcag 1201aatgaagaag atgagcccaa ggggctgggt gggtccaggg aggccagggg ctcggtgtga38. human Kir 4.1- cDNA    1atgacgtcag ttgccaaggt gtattacagt cagaccactc agacagaaag ccggccccta   61atgggcccag ggatacgacg gcggagagtc ctgacaaaag atggtcgcag caacgtgaga  121atggagcaca ttgccgacaa gcgcttcctc tacctcaagg acctgtggac aaccttcatt  181gacatgcagt ggcgctacaa gcttctgctc ttctctgcga cctttgcagg cacatggttc  241ctctttggcg tggtgtggta tctggtagct gtggcacatg gggacctgct ggagctggac  301cccccggcca accacacccc ctgtgtggta caggtgcaca cactcactgg agccttcctc  361ttctcccttg aatcccaaac caccattggc tatggcttcc gctacatcag tgaggaatgt  421ccactggcca ttgtgcttct tattgcccag ctggtgctca ccaccatcct ggaaatcttc  481atcacaggta ccttcctggc gaagattgcc cggcccaaga agcgggctga gaccattcgt  541ttcagccagc atgcagttgt ggcctcccac aatggcaagc cctgcctcat gatccgagtt  601gccaatatgc gcaaaagcct cctcattggc tgccaggtga caggaaaact gcttcagacc  661caccaaacca aggaagggga gaacatccgg ctcaaccagg tcaatgtgac tttccaagta  721gacacagcct ctgacagccc cttccttatt ctacccctta ccttctatca tgtggtagat  781gagaccagtc ccttgaaaga tctccctctt cgcagtggtg agggtgactt tgagctggtg  841ctgatcctaa gtgggacagt ggagtccacc agtgccacct gtcaggtgcg cacttcctac  901ctgccagagg agatcctttg gggctacgag ttcacacctg ccatctcact gtcagccagt  961ggtaaataca tagctgactt tagccttttt gaccaagttg tgaaagtggc ctctcctagt 1021ggcctccgtg acagcactgt acgctacgga gaccctgaaa agctcaagtt ggaggagtca 1081ttaagggagc aagctgagaa ggagggcagt gcccttagtg tgcgcatcag caatgtctga39. human Kir 4.2- cDNA    1atggatgcca ttcacatcgg catgtccagc acccccctgg tgaagcacac tgctggggct   61gggctcaagg ccaacagacc ccgcgtcatg tccaagagtg ggcacagcaa cgtgagaatt  121gacaaagtgg atggcatata cctactctac ctgcaagacc tgtggaccac agttatcgac  181atgaagtgga gatacaaact caccctgttc gotgccactt ttgtgatgac ctggttcctt  241tttggagtca tctactatgc catcgcgttt attcatgggg acttagaacc cggtgagccc  301atttcaaatc ataccccctg catcatgaaa gtggactctc tcactggggc gtttctcttt  361tccctggaat cccagacaac cattggctat ggagtccgtt ccatcacaga ggaatgtcct  421catgccatct tcctgttggt tgctcagttg gtcatcacga ccttgattga gatcttcatc  481accggaacct tcctggccaa aatcgccaga cccaaaaagc gggctgagac catcaagttc  541agccactgtg cagtcatcac caagcagaat gggaagctgt gcttggtgat tcaggtagcc  601aatatgagga agagcctctt gattcagtgc cagctctctg gcaagctcct gcagacccac  661gtcaccaagg agggggagcg gattctcctc aaccaagcca ctgtcaaatt ccacgtggac  721tcctcctctg agagcccctt cctcattctg cccatgacat tctaccatgt gctggatgag  781acgagccccc tgagagacct cacaccccaa aacctaaagg agaaggagtt tgagcttgtg  841gtcctcctca atgccactgt ggaatccacc agcgctgtct gccagagccg aacatcttat  901atcccagagg aaatctactg gggttttgag tttgtgcctg tggtatctct ctccaaaaat  961ggaaaatatg tggctgattt cagtcagttt gaacagattc ggaaaagccc agattgcaca 1021ttttactgtg cagattctga gaaacagcaa ctcgaggaga agtacaggca ggaggatcag 1081agggaaagag aactgaggac acttttatta caacagagca atgtctga40. human Kir 5.1- cDNA    1atgagctatt acggcagcag ctatcatatt atcaatgcgg acgcaaaata cccaggctac   61ccgccagagc acattatagc tgagaagaga agagcaagaa gacgattact tcacaaagat  121ggcagctgta atgtctactt caagcacatt tttggagaat ggggaagcta tgtggttgac  181atcttcacca ctcttgtgga caccaagtgg cgccatatgt ttgtgatatt ttctttatct  241tatattctct cgtggttgat atttggctct gtcttttggc tcatagcctt tcatcatggc  301gatctattaa atgatccaga catcacacct tgtgttgaca acgtccattc tttcacaggg  361gcctttttgt tctccctaga gacccaaacc accataggat atggttatcg ctgtgttact  421gaagaatgtt ctgtggccgt gctcatggtg atcctccagt ccatcttaag ttgcatcata  481aataccttta tcattggagc tgccttggcc aaaatggcaa ctgctcgaaa gagagcccaa  541accattcgtt tcagctactt tgcacttata ggtatgagag atgggaagct ttgcctcatg  601tggcgcattg gtgattttcg gccaaaccac gtggtagaag gaacagttag agcccaactt  661ctccgctata cagaagacag tgaagggagg atgacgatgg catttaaaga cctcaaatta  721gtcaacgacc aaatcatcct ggtcaccccg gtaactattg tccatgaaat tgaccatgag  781agccctctgt atgcccttga ccgCaaagca gtagccaaag ataactttga gattttggtg  841acatttatct atactggtga ttccactgga acatctcacc aatctagaag ctcctatgtt  901ccccgagaaa ttctctgggg ccataggttt aatgatgtct tggaagttaa gaggaagtat  961tacaaagtga actgcttaca gtttgaagga agtgtggaag tatatgcccc cttttgcagt 1021gccaagcaat tggactggaa agaccagcag ctccacatag aaaaagcacc accagttcga 1081gaatcctgca cgtcggacac caaggcgaga cgaaggtcat ttagtgcagt tgccattgtc 1141agcagctgtg aaaaccctga ggagaccacc acttccgcca cacatgaata tagggaaaca 1201ccttatcaga aagctctcct gactttaaac agaatctctg tagaatccca aatgtag41. human Kir 6.1- cDNA    1atgttggcca gaaagagtat catcccggag gagaatgtgc tggcgcgcat cgccgcagag   61aacctgcgca agccgcgcat ccgagaccgc ctccccaaag cccgcttcat cgccaagagc  121ggggcctgca acctggcgca taagaacatc cgtgagcaag gacgctttct acaggacatc  181ttcaccacct tggtggacct gaaatggcgc cacacgctgg tcatctttac catgtccttc  241ctctgcagct ggctgctctt cgctatcatg tggtggctgg tggcctttgc ccatggggac  301atctatgctt acatggagaa aagtggaatg gagaaaagtg gtttggagtc cactgtgtgt  361gtgactaatg tcaggtcttt cacttctgct tttctcttct ccattgaagt tcaagttacc  421attgggtttg gagggaggat gatgacagag gaatgccctt tggccatcac ggttttgatt  481ctccagaata ttgtgggttt gatcatcaat gcagtcatgt taggctgcat tttcatgaaa  541acagctcagg ctcacagaag ggcagaaact ttgattttca gccgccatgc tgtgattgcc  601gtccgaaatg gcaagctgtg cttcatgttc cgagtgggtg acctgaggaa aagcatgatc  661attagtgcct ctgtgcgcat ccaggtggtc aagaaaacaa ctacacctga aggggaggtg  721gttcctattc accaactgga cattcctgtt gataacccaa tcgagagcaa taacattttt  781ctggtggccc ctttgatcat ctgccacgtg attgacaagc gcagtcccct gtatgacatc  841tcagcaactg acctggccaa ccaagacttg gaggtcatag ttattctgga aggagtggtt  901gaaactactg gcatcaccac acaagcacga acctcctaca ttgctgagga gatccaatgg  961ggccaccgct ttgtgtccat tgtgactgag gaagaaggag tgtattctgt ggattactcc 1021aaatttggca acactgttaa agtagctgct ccacggtgca gtgcccgaga gctggatgag 1081aaaccttcca tccttattca gaccctccaa aagagtgaac tgtctcatca aaattctctg 1141aggaagcgca actccatgag aagaaacaat tccatgagga ggaacaattc tatccgaagg 1201aacaattctt ccctcatggt accaaaggtg caatttatga ctccagaagg aaatcaaaac 1261acatcggaat catga 42. human Kir 6.2- cDNA    1atgctgtccc gcaagggcat catccccgag gaatacgtgc tgacacgcct ggcagaggac   61cctgccaagc ccaggtaccg tgcccgccag cggagggccc gctttgcgtc caagaaaggc  121aactgcaacg tggcccacaa gaacatccgg gagcagggcc gcttcctgca ggacgtgttc  181accacgctgg tggacctcaa gtggccacac acattgctca tcctCaccat gtccttcctg  241tgcagctggc tgctcttcgc catggcctgg tggctcatcg ccttcgccca cggtgacctg  301gcccccagcg agggcactgc tgagccctgt gtcaccagca tccactcctt ctcgtctgcc  361ttccttttct ccattgaggt ccaagtgact attggctttg gggggcgcat ggtgactgag  421gagtgcccac tggccatcct gatcctcatc gtgcagaaca tcgtggggct catgatcaac  481gccatcatgc ttggctgcat cttcatgaag actgcccaag cccaccgcag ggctgagacc  541ctcatcttca gcaagcatgc ggtgatcgcc ctgcgccacg gccgcctctg catcatgcta  601cgtgtgggtg acctccgcaa gagcatgatc atcagcgcca ccatccacat gcaggtggta  661cgcaagacca ccagccccga gggcgaggtg gtgcccctcc accaggtgga catccccatg  721gagaacggcg tgggtggcaa cagcatcttc ctggtggccc cgctgatcat ctaccatgtc  781attgatgcca acagcccact ctacgacctg gcacccagcg acctgcacca ccaccaggac  841ctcgagatca tcgtcatcct ggaaggcgtg gtggaaacca cgggcatcac cacccaggcc  901cgcacctcct acctggccga tgagatcctg tggggccagc gctttgtgcc cattgtagct  961gaggaggacg gacgttactc tgtggactac tccaagtttg gcaacaccgt caaagtgccc 1021acaccactct gcacggcccg ccagcttgat gaggaccaca gcctactgga agctctgacc 1081ctcgcctcag cccgcgggcc cctgcgcaag cgcagcgtgc ccatggccaa ggccaagccc 1141aagttcagca tctctccaga ttccctgtcc tga 43. human Kir 7.1- cDNA    1atggacagca gtaattgcaa agttattgct cctctcctaa gtcaaagata ccggaggatg   61gtcaccaagg atggccacag cacacttcaa atggatggcg ctcaaagagg tcttgcatat  121cttcgagatg cttggggaat cctaatggac atgcgctggc gttggatgat gttggtcttt  181tctgcttctt ttgttgtcca ctggcttgtc tttgcagtgc tctggtatgt tctggctgag  241atgaatggtg atctggaact agatcatgat gccccacctg aaaaccacac tatctgtgtc  301aagtatatca ccagtttcac agctgcattc tccttctccc tggagacaca actcacaatt  361ggttatggta ccatgttccc cagcggcgac tgtccaagtg caatcgcctt acttgccata  421caaatgctcc taggcctcat gctagaggct tttatcacag gtgcttttgt ggcgaagatt  481gcccggccaa aaaatcgagc tttttcaatt cgctttactg acacagcagt agtagctcac  541atggatggca aacctaatct tatcttccaa gtggccaaca cccgacctag ccctctaacc  601agtgtccggg tctcagctgt actctatcag gaaagagaaa atggcaaact ctaccagacc  661agtgtggatt tccaccttga tggcatcagt tctgacgaat gtccattctt catctttcca  721ctaacgtact atcactccat tacaccatca agtcctctgg ctactctgct ccagcatgaa  781aatccttctc actttgaatt agtagtattc ctttcagcaa tgcaggaggg cactggagaa  841atatgccaaa ggaggacatc ctacctaccg tctgaaatca tgttacatca ctgttttgca  901tctctgttga cccgaggttc caaaggtgaa tatcaaatca agatggagaa ttttgacaag  961actgtccctg aatttccaac tcctctggtt Cctaaaagcc caaacaggac tgacctggat 1021atccacatca atggacaaag cattgacaat tttcagatct ctgaaacagg actgacagaa 1081taa 44. chicken Kir 2.1- cDNA    1atgggcagcg tgcgaaccaa ccgctacagc atcgtgtctt cggaagagga cggcatgaag   61ctggcaacca tggccgttgc caatggcttt gggaatggaa aaagtaaggt acacaccagg  121cagcagtgca ggagccgctt tgtcaaaaaa gatggccact gcaacgtcca gtttattaat  181gtgggtgaga agggacagcg atacctcgca gacatcttca ccacttgcgt ggacatccgc  241tggaggtgga tgctggttat cttctgcctg acattcatcc tctcctggct tttctttggc  301tgtgtgtttt ggttgattgc gctgttgcac ggggatctgg agaaccaaga aaataacaaa  361ccgtgtgtct cgcaagtgag cagcttcact gcagcctttc tgttctccat tgagacccag  421accacgatcg gctatggctt caggtgcgtc acagacgagt gccccattgc tgttttcatg  481gtggttttcc agtctatagt aggctgcatc attgacgcct tcatcattgg tgccgtcatg  541gcaaagatgg ctaagccaaa aaagagaaac gaaactcttg tcttcagcca caatgccgtg  601gtggccatga gagatgggaa actgtgcctg atgtggcgtg tcggaaacct gaggaaaagc  661cacttggttg aggcacacgt gcgagcacag ctcctcaagt ccaggatcac gtcagaaggg  721gagtacatcc ctttggatga aatagacatc aatgtagggt ttgacagcgg gatagaccgc  781atattcctgg tctccccaat tacaatagta cacgaaatag atgaagatag tcctttgtat  841gacttgagca aacaagacat ggacaatgct gactttgaaa ttgtagtcat tttagagggc  901atggtggaag ccactgccat gactacccag tgccgcagct catatctggc aaatgaaatc  961ctctggggcc accgctatga gcccgtactc tttgaagaaa aaaactacta caaagtggac 1021tattcaaggt tccacaaaac atacgaagtg cccaacacac ccatctgcag tgccagagac 1081ttagcagaaa agaaatacat tctctcgaac gcaaactcct tttgctacga gaacgaagtg 1141gccctcacca gcaaggagga ggacgagatc gacacggggg tgcccgagag cacaagcaca 1201gacacccacc ccgacatgga ccaccacaac caggcaggag tgcccctaga gccacggccg 1261ctgcggcgtg agtcggaaat atga 45. chicken Kir 2.2- cDNA    1atgactgcag gcagggtcaa cccttacagc atagtgtcct ccgaggaaga cggactgagg   61ttgaccacca tgccagggat taacggcttt ggcaatggga aaatccacac caggaggaaa  121tgcaggaaca ggtttgtaaa gaagaacggt cagtgcaatg tggagttcac caacatggat  181gacaagccac agaggtacat tgcagacatg ttcaccacgt gcgttgacat ccgttggagg  241tatatgctct cgctcttctc cctggcattt ctggtatcct ggttattgtt tgggctgatt  301ttctggctaa ttgcactcat tcatggagat ctagaaaacc caggtggaga tgataccttc  361aagccttgcg ttctgcagga caatggcttt gtggctgctt ttctgttctc catcgagacc  421caaacgacta ttggttatgg cttccgctgt gtgacagagg agtgcccgct cgcagtcttc  481atggtggtgg ttcagtccat cgtggggtgt ataatcgact ctttcatgat tggtgcaata  541atggcaaaga tggccaggcc caaaaaaagg gcccagacat tgcttttcag ccataatgca  601gtagtggcaa tgagagatgg aaaactctgc ctgatgtgga gagttgggaa tctccggaaa  661agccacatag tagaagccca cgtacgagct caattaatta agcccagaat cacagaagaa  721ggggagtaca tcccactcga ccaaatagac atcgacgtgg ggtttgacaa aggcttggac  781cgaatcttct tggtgtcccc cattaccatt ctccatgaga tcaacgaaga cagcccgctg  841ttcgggatca gccgccagga cttggagacg gatgactttg agattgtggt catcctcgaa  901ggcatggtag aagccaccgc gatgacgaca caagctcgga gctcctacct ggccagcgag  961atcctgtggg gccaccgctt cgagcccgtc ttgttcgagg agaaaaacca gtacaaagta 1021gactattccc acttccacaa aacatacgag gtcccgtcca caccccgctg cagcgccaag 1081gacttggtgg agaacaaatt cctgctgccc agcaccaact ccttctgcta cgagaatgag 1141ctggccttca tgagccgcga tgaggatgag gaggatgatg acagcagggg tttggacgac 1201ctgagcccag acaacaggca cgagttcgac cggcttcagg caacgatagc gttggatcag 1261aggtcatacc ggagggagtc agaaatatga 46. human Kir 1.1- turret regionHKDLPEFHPSANHTPCVENING 47. human Kir 1.2- turret regionHGDLLELDPPANHTPCVVQVHT 48. human Kir 2.1- turret regionHGDLDASKEGKACVSEVNS 49. human Kir 2.2- turret regionHGDLEPAEGRGRTPCVMQVHG 50. human Kir 2.3- turret regionHGDLEASPGVPAAGGPAAGGGGAAPVAPKPCIMHVNG 51. human Kir 2.4- turret regionHGDLAAPPPPAPCFSHVAS 52. human Kir 3.1- turret regionRGDLNKAHVGNYTPCVANVYN 53. human Kir 3.4- turret regionRGDLDHVGDQEWIPCVENLSG 54. human Kir 6.1- turret regionHGDIYAYMEKSGMEKSGLESTVCVTNVRS 55. human Kir 6.2- turret regionHGDLAPSEGTAEPCVTSIHS 56. human Kir 7.1- turret regionNGDLELDHDAPPENHTICVKYITS 57. chicken Kir 2.1- turret regionHGDLENQENNKPCVSQVSS 58. chicken Kir 2.2- turret regionHGDLENPGGDDTFKPCVLQVNG 59. Kir Bac 1.1- turret regionSPARKPPRGGRRIWSGTREVIAYGMPASVWRDLYYWALKVSWPVFFASLAALFVVNNTLFALLYQLGDAPIANQSPPGFVGAFFFSVETLATVGYGDMHPQTVYAHAIATLEIFVGMSGIALSTGLVFARFARPRAKIMFARHAIVRPFNGRMTLMVRAANARQNVIAEARAKMRLMRREHSSEGYSLMKIHDLKLVRNEHPIFLLGWNMMHVIDESSPLFGETPESLAEGRAMLLVMIEGSDETTAQVMQARHAWEHDDIRWHHRYVDLMSDVDGMTHIDYTRFNDTEPVEPPGAAPDAQAFAAKPGE 60. KcsA- turret regionMAPMLSGLLARLVKLLLGRHGSALHWRAAGAATVLLVIVLLAGSYLAVLAERGAPGAQLITYPRALWWSVETATTVGYGDLYPVTLWGRCVAVVVMVAGITSFGLV TAALATWFVGREQERRGH61. rKv1.2- turret regionMRELGLLIFFLFIGVILFSSAVYFAEADERDSQFPSIPDAFWWAVVSMTTVGYGDMVPTTIGGKIVGSLCAIAGVLTIALPVPVIVSNFNYFYHRETEGE

1. A method for identifying a compound that modulates ion channelactivity of a Kir channel comprising: identifying a compound which bindsthe turret region of a Kir channel; and determining if the compoundmodulates ion channel activity of the Kir channel.
 2. The method ofclaim 1, wherein the compound is an antibody.
 3. The method of claim 2,wherein the antibody is human, chimeric, or humanized.
 4. The method ofclaim 2, wherein the antibody is selected from the group consisting ofpolyclonal antibodies, monoclonal antibodies, an intact immunoglobulinmolecule, an antibody fragment, a scFv, a Fab, a F(ab)2, a Fv, and adisulfide linked Fv.
 5. The method of claim 1, wherein the compound is anucleic acid.
 6. The method of claim 5, wherein the nucleic acid isselected from the group consisting of DNA and RNA.
 7. The method ofclaim 5, wherein the identifying includes in vitro selection of thenucleic acid.
 8. The method of claim 1, wherein the compound is aprotein/peptide.
 9. The method of claim 8, wherein the protein/peptideis attached to a protein scaffold.
 10. The method of claim 8, whereinthe protein/peptide is displayed on the surface of a phage.
 11. Themethod of claim 1, wherein the compound is a small molecule.
 12. Themethod of claim 1, wherein the Kir channel is human.
 13. The method ofclaim 1, wherein the Kir channel is a chicken/human hybrid.
 14. Themethod of claim 13, wherein the chicken/human hybrid Kir channelcomprises a human Kir channel turret region.
 15. The method of claim 1,wherein the identifying step comprises an ELISA and a Western blot todetermine if the compound binds to a properly folded Kir channel but notto a denatured Kir channel.
 16. The method of claim 1, wherein theidentifying step comprises determining if the compound binds to a Kirchannel with a normal turret region but not a mutated turret region. 17.The method of claim 1 wherein the determining step comprises anelectrophysiological assay.
 18. The method of claim 17, wherein theelectrophysiological assay is selected from the group consisting oftwo-electrode voltage clamp, patch clamp, and planar lipid bilayerassays.
 19. The method of claim 1, wherein the determining stepcomprises a fluorescent assay.
 20. The method of claim 19, wherein thefluorescent assay utilizes a thallium specific fluorescent dye.
 21. Amethod for identifying a compound that selectively modulates ion channelactivity of a specific type of Kir channel comprising: identifying acompound which binds the turret region of a specific type of Kir channelbut does not bind to other types of Kir channels; and determining if thecompound modulates the activity of the Kir channel.
 22. The method ofclaim 21, wherein the identifying comprises deter pining if the compoundbinds the turret region of the specific type of Kir channel but does notbind the turret region of other type of Kir channels.
 23. A method ofidentifying a compound to treat a condition associated with abnormal ionchannel activity by a Kir channel comprising: identifying a compoundwhich binds the turret region of a Kir channel; determining if saidcompound modulates ion channel activity of the Kir channel; andadministering said compound which modulates ion channel activity to asubject to determine if the compound is able to treat said condition.24. The method of claim 23, wherein the condition is selected from thegroup consisting of diabetes mellitus, hypertension, cardiac arrhythmia,and epilepsy. 25.-43. (canceled)